Guanine riboswitch binding compounds and their use as antibiotics

ABSTRACT

The present invention includes novel compounds and pharmaceutically acceptable formulations of said compounds which exhibit antibiotic activity against microorganisms bearing a guanine riboswitch that controls the expression of the guaA gene, including organisms which are resistant to certain antibiotic families, and which are useful as antibacterial agents for treatment or prophylaxis of bacterial infections in animals or in humans, in particular but not limited to infections of the mammary gland, or their use as antiseptics, agents for sterilization or disinfection.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a PCT application filed on Jun. 14, 2010 andpublished in English under PCT Article 21(2), claiming benefit of U.S.Provisional applications Ser. No. 61/186,669, filed on Jun. 12, 2009 andNo. 61/307,088 filed on Feb. 23, 2010. All documents above areincorporated herein in their entirety by reference.

FIELD OF THE INVENTION

The present invention relates to novel antimicrobial compounds. Morespecifically, the present invention is concerned with novelantimicrobial compounds which bind to a guanine riboswitch and inhibitthe expression of the microbial gene guaA, methods of manufacturingsame, disinfection, sterilization or antisepsis methods using same, andmethods of treating or preventing microbial infections involving theadministration of same.

BACKGROUND OF THE INVENTION

Bovine mastitis is the most frequently occurring and costly diseaseaffecting dairy producers. The transmittable bacterium Staphylococcusaureus is the most common cause of bovine mastitis and currentantibiotic therapies usually fail to eliminate the infection from dairyherds (Sears, P. M. and K. K. McCarthy, 2003).

Multiple drug resistance (MDR), partly due to the excessive and improperuse of antibiotics both in human medicine and food animal production, isa growing problem that has come to the forefront particularly in thelast decade. A major cause of this widespread multi-drug resistance isthe fact that recent drug design has been largely based on a limitednumber of new chemical scaffolds, allowing pathogens to adapt andcircumvent common antibiotic action mechanisms (Blount and Breaker,2006). Staphylococcus aureus and Clostridium difficile, which areparticularly prone to developing antibiotic resistance, are nosocomialpathogens of major importance responsible for a significant mortalityrate in hospitals and increased health care costs (Talbot et al, 2006).Alternative antibacterial drugs targeting RNA, mainly based on afortuitous interaction between an exogenous ligand and its RNA target,have recently been developed and may help alleviate the problem of MDR(Knowles et al, 2002). However, there remains a great need for theidentification of novel antimicrobial targets and compounds.

Bacterial riboswitches as a mechanism for regulating gene expression areknown in the art. Riboswitches are segments of the 5′-untranslatedregion of certain mRNA molecules that, upon recognition of specificligands, modify the expression of one or more proteins encoded in themessage. Ligand-binding results in structural changes in the riboswitchthat affect the ability of the mRNA molecule to be properly transcribedor translated. Thus riboswitches can function as RNA sensors, allowingcontrol of gene expression at the mRNA level. The best-characterizedriboswitches contain an aptamer region that is involved in ligandbinding and an expression platform that is responsible for bringingabout changes in gene expression (Coppins et al., 2007).

The scientific literature suggests that all bacterial riboswitchesindiscriminately represent molecular antimicrobial targets. However, inorder to develop novel specific antimicrobial therapies that will avoidcontributing to widespread MDR, it would be highly desirable to identifyriboswitches that are therapeutically selective, i.e., riboswitches thatdo not represent antimicrobial targets in all microbial species orcommensal microorganisms that bear similar switches but on the contrary,riboswitches that have specific properties that only appear in thetargeted pathogens.

Guanine riboswitches represent a subclass of riboswitches that are knownin the art (Barrick and Breaker, 2007; Batey et al, 2004; Mulhbacher andLafontaine, 2007). Application WO 2006/55351 teaches that the guanineriboswitch, in the presence of the guanine ligand, inhibits theexpression of the genes xpt and pbuX in the non-pathogenic bacteriaBacillus subtilis. Some of the guanine analogs proposed in applicationWO 2006/55351 as potential therapeutic agents were reported to havenon-specific antibacterial activities in vitro against a broad selectionof pathogenic and non-pathogenic bacteria including the non-pathogenicBacillus subtilis. Furthermore, later studies from the sameinvestigators reported that these two genes (xpt and pbuX), assumed tobe controlled by the guanine riboswitch, were expected to be nonessential for the survival or virulence of Staphylococcus aureus (Blountand Breaker, 2006). It is well known in the field that genes (and theirproducts) that are essential for the survival or virulence of amicrobial pathogen represent ideal targets to obtain quick microbialkilling, while avoiding the development of microbial resistance arisingfrom mutations inactivating non-essential anti-microbial targets. Theteachings of WO 2006/55351 and of Blount and Breaker (2006) thussuggested that the guanine riboswitch was not a desirable target forselective antibacterial intervention against the pathogen S. aureus invivo.

Hence in this field of research and therapeutic applications, it wouldbe highly desirable to identify microbial pathogens having essentialsurvival or virulence gene(s) under control of a riboswitch. It wouldalso be highly desirable to identify antimicrobial compounds thatspecifically bind to these riboswitches and selectively inhibit theexpression of the gene(s) essential for the survival or virulence ofspecific pathogens in vivo. Such compounds would allow selectivetreatment of pathogens affecting animals and humans. It would also behighly desirable to identify compounds that are sufficiently distinctfrom the natural riboswitch ligand to avoid transformation of thecompound by the host or microbial flora (for example by ribosylation)and to prevent potential broad and non-specific bacterial growthinhibition or toxicity to the mammalian host.

The present invention seeks to meet these and other needs.

The present description refers to a number of documents, the content ofwhich is herein incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

The present invention discloses that expression of the gene guaA(Tiedeman et al, 1985)encoding GMP synthetase (glutamineaminotransferase), which catalyses the synthesis of GMP(guanosine-5′-monophosphate) is under the control of a guanineriboswitch and is needed for survival or virulence in some relevantenvironments (e.g., during infection of a mammalian host) for specificpathogens. The present invention also presents antibiotic-like compoundswith antimicrobial activity against such pathogens. Furthermore, suchcompounds cannot be modified by ribosylation to prevent incorporationinto cellular nucleosides, nucleotides or nucleic acids, and thusprevent broad, non-specific toxicity for bacterial species not targetedby the compound and for the mammalian or animal host in which thepresent compounds are used for therapeutic intervention.

More specifically, in accordance with an aspect of the presentinvention, there is provided a compound of the general formula 1.0,

wherein, when said compound is bound to a guanine riboswitch, R3 mayserve as a hydrogen bond acceptor but cannot be ribosylated; wherein

represents a single or double bond; wherein R3 is —N═, —S—, —CH— or —O—;wherein R6 is a carbon atom; wherein R1, R2, R4 or R5 are identical ordifferent and are independently —NR8-, —CHR8-, ═CR8-, —C(═O)— or'C(═NR8)-; wherein R8 is —H, —NH₂, —OH, —SH, a halide, fluorine,chlorine, bromine, iodine, —CO₂H, —CO₂-alkyl, —CO₂-aryl, —C(O)NH₂, —CH₃,—POOH, —SO₂alkyl, —SO₂aryl, —SH, substituted or unsubstituted alkyl,substituted or unsubstituted alkoxy, substituted or unsubstituted aryl,substituted or unsubstituted aryloxy, substituted or unsubstitutedbenzyloxy, substituted or unsubstituted —NHalkyl, substituted orunsubstituted —NHalkoxy, substituted or unsubstituted —NHC(O)alkyl,substituted or unsubstituted —NHCO₂alkyl, substituted or unsubstituted—NH—NHalkyl, substituted or unsubstituted —NH—NHalkoxy, substituted orunsubstituted —NH—SO₂alkyl, —NHC(O)NH₂, —NH—NH₂, —NH—SO₂—R9,—NHCO₂CH₂—R9, —NH—OR9, —NH₂—R9, —NH—NH—R9, —NHR9, —NH—CH₂—R9, or—NH—NH'CH₂—R9, wherein R9 is:

wherein n is an integer from 1 to 5; wherein R10 is —H, —NH₂, —OH,alkoxy, —N-morpholino, a halide, fluorine, chlorine, bromine or iodine;wherein, R7 is ═O, —OH, —SH, —NH₂, —CO₂H, —CO₂-alkyl, —CO₂-aryl,—C(O)NH₂, -alkoxy, -aryloxy, -benzyloxy, a halide, fluorine, chlorine,bromine, iodine, —NHalkyl, —NHalkoxy, —NHC(O)alkyl, —NHCO₂alkyl, ═NR8,═NR9, —NHCO₂CH₂—R9, —NHC(O)NH₂, —NH—NH₂, —NH—NHalkyl, —NH—NHalkoxy,—SO₂alkyl, —SO₂aryl, —NH—SO₂alkyl, —NH—SO₂—R9, —NH—OR9, —NH—R9,—NH—NH—R9, —NH—NH—CH₂—R9, or —NH—CH₂—R9, wherein R8 and R9 are asdefined above, with the proviso that the compound is not:4-hydroxy-2,5,6-triaminopyrimidine (1.01);2,4-diamino-6-hydroxypyrimidine (1.13); or4,5-diamino-6-hydroxy-2-mercaptopyrimidine (1.16).

It is well understood by those skilled in the art that heterocycliccompounds such as the compounds of the present invention can exist underdifferent tautomeric forms, each tautomeric form being described using aspecific formula and a specific name. Herein, the use of the name of onetautomeric form of a compound is meant to refer to and encompass allother tautomeric forms of the same compound. For example, as usedherein, the terms “4-hydroxy-2,5,6-triaminopyrimidine” and “formula1.01” are meant to refer to both “4-hydroxy-2,5,6-triaminopyrimidine”and “2,5,6-triaminopyrimidine-4-one”.

As used herein, the terms “4,5-diamino-6-hydroxy-2-mercaptopyrimidine”or “formula 1.16” are meant to refer to both“4,5-diamino-6-hydroxy-2-mercaptopyrimidine” and“5,6-diamino-2-mercaptopyrimidine-4-one”.

As used herein, the terms “2,4-diamino-6-hydroxypyrimidine” or “formula1.13” are meant to refer to both “2,4-diamino-6-hydroxypyrimidine” and“2,6-diaminopyrimidine-4-one”.

As used herein, the terms “9-oxoguanine” or “formula 2.02” are meant torefer to both “9-oxoguanine” and“5-Amino-6H-oxazolo[5,4-d]pyrimidin-7-one”.

In a specific embodiment of the composition of the general formula 1.0,(i) R1 is —NH—; (ii) R2 is —C(NH₂)═ or ═C(SH)—; (iii) R3 is —N═; (iv) R4is ═CNH₂—; R5 is —CH═ or 'C(NH₂)═; R6 is —C═; R7 is ═ NH or ═O; or anycombination of (i) to (vii).

In accordance with another aspect of the present invention, there isprovided a compound of the general formula 2.0,

wherein, when said compound is bound to a guanine riboswitch, R9 mayserve as a hydrogen bond donor but cannot be ribosylated; wherein

represents a single or double bond; wherein R3 is —N═, —S—, or —O—;wherein R1 or R2 are identical or different and are independently—NR11-, —CHR11-, ═CR11-, —C(═O)— or —C(═NR11)-; wherein R11 is —H, —NH₂,—OH, —SH, —CO₂H, —CO₂-alkyl, —CO₂-aryl, —C(O)NH₂, —CH₃, —POOH,—SO₂alkyl, —SO₂aryl, —SH, substituted or unsubstituted alkyl,substituted or unsubstituted alkoxy, substituted or unsubstituted aryl,substituted or unsubstituted aryloxy, substituted or unsubstitutedbenzyloxy, substituted or unsubstituted —NHalkyl, substituted orunsubstituted —NHalkoxy, substituted or unsubstituted —NHC(O)alkyl,substituted or unsubstituted —NHCO₂alkyl, substituted or unsubstituted—NH—NHalkyl, substituted or unsubstituted —NH—NHalkoxy, substituted orunsubstituted —NH—SO₂alkyl, —NHC(O)N H₂, —NH—NH₂, —NH—SO₂—R12,—NHCO₂CH₂—R12, —NH—OR12, —NH₂—R12, —NH—NH—R12, —NHR12, —NH—CH₂—R12, or—NH—NH—CH₂—R12;

wherein R12 is:

wherein n is in integer from 1 to 5; wherein R13 is at least one of —H,—NH₂, —OH, alkoxy, —N-morpholino, a halide, fluorine, chlorine, bromine,iodine; wherein R4, R5 and R6 are carbon atoms; wherein R7 is —═N—,—NH—, —CH₂—; —O— or —S—; wherein R8 is —CH₂, —O—, —S—, —CHR13- or—CR13=, wherein R13 is as defined above; wherein R9 is —CH₂—; —O—, —S—,or —P(OOH)—, —N═; wherein R10 is ═O, —OH, —SH, —NH₂, —CO₂H, —CO₂-alkyl,—CH₂-aryl, —C(O)NH₂, -alkoxy, -aryloxy, -benzyloxy, a halide, fluorine,chlorine, bromine, iodine, —NHalkyl, —NHalkoxy, - NHC(O)alkyl,—NHCO₂alkyl, ═NR11, =NR12, —NHCO₂CH₂—R12, —NHC(O)NH₂, —NH—NH₂,—NH—NHalkyl, —NH—NHalkoxy, —SO₂alkyl, —SO₂aryl, —NH—SO₂alkyl,—NH—SO₂—R12, —NH—R12, —NH—R12, —NH—NH—R12, —NH—NH—CH₂—R12, or—NH—CH₂—R12; wherein R11 and R12 are as defined above, with the provisothat the compound is not: guanine, hypoxanthine, xanthine or5-amino-2-chloro-2,3-dihydrothiazolo[4,5]pyrimidine-7-(6H)-one (2.17).

In accordance with another aspect of the present invention, there isprovided a compound of the general formula 3.0,

wherein, when the compound is bound to a guanine riboswitch, R10 mayserve as a hydrogen bond donor but cannot be ribosylated; wherein

represents a single or double bond; wherein R3 is —N═, —S—, or —O—;wherein R10 is —N═, —CH₂—; —O—, —S—, or —P(OOH)—; wherein R1, R2 andR9are identical or different and are independently —NR12-, —CHR12-,═OR12-, —O(═O)— or—O(═NR12)—; wherein R12 is —H, —NH₂, —OH, —SH, —CO₂H,—CO₂-alkyl, —CO₂-aryl, —C(O)NH₂, —CH₃, —POOH, —SO₂alkyl, —SO₂aryl, —SH,substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy,substituted or unsubstituted aryl, substituted or unsubstituted aryloxy,substituted or unsubstituted benzyloxy, substituted or unsubstituted—NHalkyl, substituted or unsubstituted —NHalkoxy, substituted orunsubstituted —NHC(O)alkyl, substituted or unsubstituted —NHCO₂alkyl,substituted or unsubstituted —NH—NHalkyl, substituted or unsubstituted—NH—NHalkoxy, substituted or unsubstituted —NH—SO₂alkyl, —NHC(O)NH₂,—NH—NH₂, —NH—SO₂—R13,—NHCO₂CH₂—R13, —NH—OR13, —N+H2-R13, —NH—NH—R13,—NHR13, —NH—CH₂—R13, or —NH—NH—CH₂—R13;

wherein R13 is:

wherein n is an integer from 1 to 5; wherein R14 is —H, —NH₂, —OH,alkoxy, —N-morpholino, a halide, fluorine, chlorine, bromine, or iodine;wherein R4, R5 and R6 are carbon atoms; wherein R7 and R8 are identicalor different and are independently —CH═N—, —CH₂—O—, CH₂—S—, or CH₂SO₂—;wherein R11 is ═O, —OH, —SH, —NH₂, —CO₂H, —CO₂-alkyl, —CO₂-aryl,'C(O)NH₂, -alkoxy, -aryloxy, -benzyloxy, a halide, fluorine, chlorine,bromine, iodine, —NHalkyl, —NHalkoxy, —NHC(O)alkyl, —NHCO₂alkyl, ═NR12,═NR13, —NHCO₂CH₂—R13, —NHC(O)NH₂, —NH—NH₂, —NH—NHalkyl, —NH—NHalkoxy,—SO₂alkyl, —SO₂aryl, —NH—SO₂alkyl, —NH—SO₂—R13, —NH—OR13, —NH—R13,—NH—NH—R13, —NH—NH—CH₂—R13, or —NH—CH₂—R13; and wherein R12 and R13 areas defined above.

In accordance with an aspect of the present invention, there is provideda compound of the general formula (2.0a),

wherein R2, R7, R8, R9 and R10 are as defined for formula 2.0 above.

In a specific embodiment of the compound of the present invention, thereis provided a compound of the general formula (2.0), wherein: (i) R1 is—NH—; (ii) R2 is ═CNH₂—; (iii) R3 is —N═; (iv) R7 is —N═ or —S—; (v) R8is —CR13=; (vi) R9 is —O— or —N═; (vii) R10 is ═O or ═NH; or (viii) anycombination of (i) to (vii).

In another specific embodiment of the compound of formula 2.0a of thepresent invention, R2 is ═CNH₂, in another specific embodiment, R7 is—N═ or —S—, in another specific embodiment, R8 is —CR13=, in anotherspecific embodiment, R9 is —O— or —N═, in another specific embodiment,R10 is ═O or ═NH.

In accordance with an aspect of the present invention, there is provideda compound of the general formula 3.0a,

wherein, R2, R7, R10 and R11 are as defined as defined for formula 3.0above.

In accordance with another aspect of the present invention, there isprovided a compound of the general formula 1.0a,

wherein R2 and R7 are as defined for formula 1.0 above and wherein R5 is—CH═ or —C(NH₂)═ while R4 is —C(NH₂)═.

In a specific embodiment of the compound of the general formula 1.0a, R7is ═O or ═NH. In a specific embodiment of the compound of the generalformula 1.0a, R2 is —C(NH₂)═ or —C(SH)═.

In accordance with another aspect of the present invention, there isprovided the compound defined above, for (a) preventing or treating amicrobial infection in a subject, wherein said microbial infection iscaused by a pathogen bearing a guanine riboswitch that controls theexpression of guaA; or (b) the disinfection, sterilization and/orantisepsis of an object from a pathogen bearing a guanine riboswitchthat controls the expression of guaA; or (c) treating a multi-drugresistant bacteria or preventing the development of a multi-drugresistant bacteria, wherein the bacteria bears a guanine riboswitch thatcontrols the expression of guaA.

In accordance with another aspect of the present invention, there isprovided a composition comprising a compound of the general formula 1.0,

wherein, when said compound is bound to a guanine riboswitch, R3 mayserve as a hydrogen bond acceptor but cannot be ribosylated; wherein

represents a single or double bond; wherein R3 is —N═, —S—, —CH— or —O—;wherein R6 is a carbon atom; wherein R1, R2, R4 or R5 are identical ordifferent and are independently —NR8-, —CHR8-, ═CR8-, —O(═O)— or—C(═NR8)-; wherein R8 is —H, —NH₂, —OH, —SH, a halide, fluorine,chlorine, bromine, iodine, —CO₂H, —CO₂-alkyl, —CO₂-aryl, —C(O)NH₂, —CH₃,—POOH, —SO₂alkyl, —SO₂aryl, —SH, substituted or unsubstituted alkyl,substituted or unsubstituted alkoxy, substituted or unsubstituted aryl,substituted or unsubstituted aryloxy, substituted or unsubstitutedbenzyloxy, substituted or unsubstituted —NHalkyl, substituted orunsubstituted —NHalkoxy, substituted or unsubstituted —NHC(O)alkyl,substituted or unsubstituted —NHCO₂alkyl, substituted or unsubstituted—NH—NHalkyl, substituted or unsubstituted —NH—NHalkoxy, substituted orunsubstituted —NH—SO₂alkyl, —NHC(O)NH₂, —NH—NH₂, —NH—SO₂—R9,—NHCO₂CH₂—R9, —NH—OR9, —NH₂—R9, —NH—NH—R9, —NHR9, —NH—CH₂—R9, or—NH—NH—CH₂—R9, wherein R9 is:

wherein n is an integer from 1 to 5; wherein R10 is -H, —NH₂, —OH,alkoxy, —N-morpholino, a halide, fluorine, chlorine, bromine or iodine;wherein, R7 is ═O, —OH, —SH, —NH₂, —CO₂H, —CO₂-alkyl, —CO₂-aryl,—C(O)NH₂, -alkoxy, -aryloxy, -benzyloxy, a halide, fluorine, chlorine,bromine, iodine, —NHalkyl, —NHalkoxy, —NHC(O)alkyl, —NHCO₂alkyl, ═NR8,═NR9, —NHCO₂CH₂—R9, —NHC(O)NH₂, —NH—NH₂, —NH—NHalkyl, —NH—NHalkoxy,—SO₂alkyl, —SO₂aryl, —NH—SO₂alkyl, —NH—SO₂—R9, —NH—OR9, —NH—R9,—NH—NH—R9, —NH—NH—CH₂—R9, or —NH—CH₂—R9, wherein R8 and R9 are asdefined above, and (a) an antibiotic; (b) an antiseptic; (c) adisinfectant; (d) a diluent; (e) an excipient; (f) a pharmaceuticallyacceptable carrier; or any combination of (a)-(f).

In accordance with another aspect of the present invention, there isprovided a composition comprising a compound of the general formula 2.0,

wherein, when said compound is bound to a guanine riboswitch, R9 mayserve as a hydrogen bond donor but cannot be ribosylated; wherein

represents a single or double bond; wherein R3 is —N═, —S—, or —O—;wherein R1 or R2 are identical or different and are independently—NR11-, —CHR11-, ═CR11-, —O(═O)— or —C(═NR11)-; wherein R11 is —H, —NH₂,—OH, —SH, —CO₂H, —CO₂-alkyl, —CO₂-aryl, —C(O)NH₂, —CH₃, —POOH,—SO₂alkyl, —SO₂aryl, —SH, substituted or unsubstituted alkyl,substituted or unsubstituted alkoxy, substituted or unsubstituted aryl,substituted or unsubstituted aryloxy, substituted or unsubstitutedbenzyloxy, substituted or unsubstituted —NHalkyl, substituted orunsubstituted —NHalkoxy, substituted or unsubstituted —NHC(O)alkyl,substituted or unsubstituted —NHCO₂alkyl, substituted or unsubstituted—NH—NHalkyl, substituted or unsubstituted —NH—NHalkoxy, substituted orunsubstituted —NH—SO₂alkyl, —NHC(O)N H₂, —NH—NH₂, —NH—SO₂—R12,—NHCO₂CH₂—R12, —NH—OR12, —NH₂—R12, —NH—NH—R12, —NHR12, —NH—CH₂—R12, or—NH—NH—CH₂—R12;

wherein R12 is:

wherein n is in integer from 1 to 5; wherein R13 is at least one of —H,—NH₂, —OH, alkoxy, —N-morpholino, a halide, fluorine, chlorine, bromine,iodine; wherein R4, R5 and R6 are carbon atoms; wherein R7 is —═N—,—NH—, —CH₂—; —O— or —S—; wherein R8 is —CH₂, —O—, —S—, —CHR13- or—OR13=,wherein R13 is as defined above; wherein R9 is —CH₂—; —O—, —S—, or—P(OOH)—, —N═; wherein R10 is ═O, —OH, —SH, —NH₂, —CO₂H, —CO₂-alkyl,—CO₂-aryl, —C(O)NH₂, -alkoxy, -aryloxy, -benzyloxy, a halide, fluorine,chlorine, bromine, iodine, —NHalkyl, —NHalkoxy, —NHC(O)alkyl,—NHCO₂alkyl, ═NR11, ═NR12, —NHCO₂CH₂—R12, —NHC(O)NH₂, —NH—NH₂,—NH—NHalkyl, —NH—NHalkoxy, —SO₂alkyl, —SO₂aryl, —NH—SO₂alkyl,—NH—SO₂—R12, —NH—R12, —NH—R12, —NH—NH—R12, —NH—NH—CH₂—R12, or—NH—CH₂—R12; wherein R11 and R12 are as defined above, and an (a)antibiotic; (b) an antiseptic; (c) a disinfectant; (d) a diluent; (e) anexcipient; (f) a pharmaceutically acceptable carrier; or any combinationof (a)- (f).

In accordance with another aspect of the present invention, there isprovided a composition comprising a compound of the general formula 3.0,

wherein, when the compound is bound to a guanine riboswitch, R10 mayserve as a hydrogen bond donor but cannot be ribosylated; wherein

represents a single or double bond; wherein R3 is —N═, —S—, or —O—;wherein R10 is —N═, —CH₂—; —O—, —S—, or —P(OOH)—; wherein R1, R2 andR9are identical or different and are independently —NR12-, —CHR12-,═CR12-, —C(═O)— or —O(═NR12)-;

wherein R12 is —H, —NH₂, —OH, —SH, —CO₂H, —CO₂-alkyl, —CO₂-aryl,—C(O)NH₂, —CH₃, —POOH, —SO₂alkyl, —SO₂aryl, —SH, substituted orunsubstituted alkyl, substituted or unsubstituted alkoxy, substituted orunsubstituted aryl, substituted or unsubstituted aryloxy, substituted orunsubstituted benzyloxy, substituted or unsubstituted —NHalkyl,substituted or unsubstituted —NHalkoxy, substituted or unsubstituted—NHC(O)alkyl, substituted or unsubstituted —NHCO₂alkyl, substituted orunsubstituted —NH—NHalkyl, substituted or unsubstituted —NH—NHalkoxy,substituted or unsubstituted —NH—SO₂alkyl, —NHC(O)N H₂, —NH—NH₂,—NH—SO₂—R13,—NHCO₂CH₂—R13, —NH—OR13, —N+H2-R13, —NH—NH—R13, —NHR13,—NH—CH₂—R13, or —NH—NH—CH₂—R13;

wherein R13 is:

wherein n is an integer from 1 to 5; wherein R14 is —H, —NH₂, —OH,alkoxy, —N-morpholino, a halide, fluorine, chlorine, bromine, or iodine;wherein R4, R5 and R6 are carbon atoms; wherein R7 and R8 are identicalor different and are independently —CH═N—, —CH₂—O—, CH₂—S—, or CH₂SO₂—;wherein R11 is ═O, —OH, —SH, —NH₂, —CO₂H, —CO₂-alkyl, —CO₂-aryl,—C(O)NH₂, -alkoxy, -aryloxy, -benzyloxy, a halideo, fluorine, chlorine,bromine, iodine, —NHalkyl, —NHalkoxy, —NHC(O)alkyl, —NHCO₂alkyl, ═NR12,═NR13, —NHCO₂CH₂—R13, —NHC(O)NH₂, —NH—NH₂, —NH—NHalkyl, —NH—NHalkoxy,—SO₂alkyl, —SO₂aryl, —NH—SO₂alkyl, —NH—SO₂—R13, —NH—OR13, —NH—R13,—NH—NH—R13, —NH—NH—CH₂—R13, or —NH—CH₂—R13; and wherein R12 and R13 areas defined above, and (a) an antibiotic; (b) an antiseptic; (c) adisinfectant; (d) a diluent; (e) an excipient; (f) a pharmaceuticallyacceptable carrier; or any combination of (a)-(f).

In a specific embodiment of the composition of the present invention,the compound is of the general formula,

In another specific embodiment of the composition of the presentinvention, the compound is of the general formula,

In another specific embodiment of the composition of the presentinvention, the compound is of the general formula,

wherein R5 and R4 are independently —CH═ or —C(NH₂)═.

In accordance with another aspect of the present invention, there isprovided a composition comprising (i) 4-hydroxy-2,5,6-triaminopyrimidine(formula 1.01); (ii) 4,5-diamino-6-hydroxy-2-mercaptopyrimidine (formula1.16); (iii) 2,4-diamino-6-hydroxypyrimidine (formula 1.13); or (iv)5-amino-2-chloro-2,3-dihydrothiazolo[4,5]pyrimidine-7-(6H)-one (2.17);and (a) an antibiotic; (b) an antiseptic; (c) a disinfectant; (d) apharmaceutically acceptable carrier; or (e) any combination of (a)-(d).

In accordance with another aspect of the present invention, thecomposition defined above is for (a) preventing or treating a microbialinfection in a subject, wherein said microbial infection is caused by apathogen bearing a guanine riboswitch that controls the expression ofguaA; or (b) the disinfection, sterilization and/or antisepsis of anobject from a pathogen bearing a guanine riboswitch that controls theexpression of guaA; or (c) treating a multi-drug resistant bacteria orpreventing the development of a multi-drug resistant bacteria, whereinthe bacteria bears a guanine riboswitch that controls the expression ofguaA.

In another specific embodiment, said composition is a pharmaceuticalcomposition.

In accordance with another aspect of the present invention there isprovided a method of preventing or treating a microbial infection in asubject, said method comprising administering to said subject atherapeutically effective amount of a non ribosylable ligand of aguanine riboswitch, wherein said microbial infection is caused by apathogen bearing the guanine riboswitch, and wherein the guanineriboswitch controls the expression of guaA.

In accordance with another aspect of the present invention, there isprovided a method of preventing or treating a microbial infection in asubject, said method comprising administering to said subject atherapeutically effective amount of the compound defined above or of theabove-mentioned composition, wherein said microbial infection is causedby a pathogen bearing a guanine riboswitch that controls the expressionof guaA.

In a specific embodiment of the method, said non ribosylable ligand of aguanine riboswitch is (i) the compound defined above; (ii)4-hydroxy-2,5,6-triaminopyrimidine (formula 1.01); (iii)4,5-diamino-6-hydroxy-2-mercaptopyrimidine (formula 1.16); (iv)2,4-diamino-6-hydroxypyrimidine (formula 1.13); or (v)5-amino-2-chloro-2,3-dihydrothiazolo[4,5]pyrimidine-7-(6H)-one (2.17).

In a specific embodiment of the method of the present invention, saidsubject is an animal (e.g., cattle such as cow; goat, ewe, ass, horse,pig; cat; dog; etc.). n another specific embodiment, said subject is acow. In another specific embodiment, said subject is a human. In anotherspecific embodiment, said pathogen is a bacteria belonging to the genusStaphylococcus or Clostridium. In another specific embodiment, saidbacteria is Staphylococcus aureus, methicillin-resistant Staphylococcusaureus, Staphylococcus epidermidis, Staphylococcus haemolyticus,Clostridium botulinum or Clostridium difficile. In another specificembodiment, said infection is a mammary gland infection.

In accordance with another aspect of the present invention, there isprovided a use of a therapeutically effective amount of a nonribosylable ligand of a guanine riboswitch, for preventing or treating amicrobial infection in a subject, wherein said microbial infection iscaused by a pathogen bearing the guanine riboswitch, and wherein theguanine riboswitch controls the expression of guaA.

In accordance with another aspect of the present invention, there isprovided a use of a therapeutically effective amount of a nonribosylable ligand of a guanine riboswitch, for the manufacture of amedicament for preventing or treating a microbial infection in asubject, wherein said microbial infection is caused by a pathogenbearing the guanine riboswitch, and wherein the guanine riboswitchcontrols the expression of guaA.

In accordance with another aspect of the present invention, there isprovided a use of the compound defined above or of the above-mentionedcomposition, for preventing or treating a microbial infection in asubject, wherein said microbial infection is caused by a pathogenbearing the guanine riboswitch, and wherein the guanine riboswitchcontrols the expression of guaA.

In accordance with another aspect of the present invention, there isprovided a use of the compound defined above or of the above-mentionedcomposition, for the manufacture of a medicament for preventing ortreating a microbial infection in a subject, wherein said microbialinfection is caused by a pathogen bearing the guanine riboswitch, andwherein the guanine riboswitch controls the expression of guaA.

In a specific embodiment of said uses, said non ribosylable ligand of aguanine riboswitch is (i) the compound defined above; (ii)4-hydroxy-2,5,6-triaminopyrimidine (formula 1.01); (iii)4,5-diamino-6-hydroxy-2-mercaptopyrimidine (formula 1.16); (iv)2,4-diamino-6-hydroxypyrimidine (formula 1.13); or (v)5-amino-2-chloro-2,3-dihydrothiazolo[4,5]pyrimidine-7-(6H)-one (2.17).

In a specific embodiment of the use of the present invention, saidsubject is an animal (e.g., cattle such as cow; goat, ewe, ass, horse,pig; cat; dog; etc.). n another specific embodiment, said subject is acow. In another specific embodiment said subject is a human. In anotherspecific embodiment, said pathogen is a bacteria belonging to the genusStaphylococcus or Clostridium. In another specific embodiment, saidbacteria is Staphylococcus aureus, methicillin-resistant Staphylococcusaureus, Staphylococcus epidermidis, Staphylococcus haemolyticus,Clostridium botulinum or Clostridium difficile. In another specificembodiment, said infection is a mammary gland infection.

In accordance with another aspect of the present invention, there isprovided a method of disinfecting and/or sterilizing an object of apathogen, said method comprising applying an effective amount of a nonribosylable ligand of a guanine riboswitch to said object, wherein saidpathogen bears a guanine riboswitch that controls the expression ofguaA.

In accordance with another aspect of the present invention, there isprovided a method of disinfecting and/or sterilizing an object of apathogen, said method comprising applying an effective amount of thecompound defined above or of the above-mentioned composition to saidobject, wherein said pathogen bears a guanine riboswitch that controlsthe expression of guaA.

In a specific embodiment of the method of the present invention, saidnon ribosylable ligand of a guanine riboswitch is (i) the compound ofdefined above; (ii) 4-hydroxy-2,5,6-triaminopyrimidine (formula 1.01);(iii) 4,5-diamino-6-hydroxy-2-mercaptopyrimidine (formula 1.16); (iv)2,4-diamino-6-hydroxypyrimidine (formula 1.13); or (v)5-amino-2-chloro-2,3-dihydrothiazolo[4,5]pyrimidine-7-(6H)-one (2.17).

In a specific embodiment of the methods, said object is an animal ormilk.

In accordance with another aspect of the present invention, there isprovided a use of an effective amount of a non ribosylable ligand of aguanine riboswitch for the disinfection, sterilization and/or antisepsisof an object from a pathogen, said pathogen bearing a guanine riboswitchthat controls the expression of guaA.

In accordance with another aspect of the present invention, there isprovided a use of the compound defined above or of the above-mentionedcomposition for the disinfection, sterilization and/or antisepsis of anobject from a pathogen bearing a guanine riboswitch that controls theexpression of guaA.

In a specific embodiment of the use, said non ribosylable ligand of aguanine riboswitch is (i) the compound defined above; (ii)4-hydroxy-2,5,6-triaminopyrimidine (formula 1.01); (iii)4,5-diamino-6-hydroxy-2-mercaptopyrimidine (formula 1.16); (iv)2,4-diamino-6-hydroxypyrimidine(formula 1.13); or (v)5-amino-2-chloro-2,3-dihydrothiazolo[4,5]pyrimidine-7-(6H)-one (2.17).

In a specific embodiment of the uses, said object is an animal or milk.

In accordance with another aspect of the present invention, there isprovided a method of selecting a pathogen treatable by a non ribosylableligand of a guanine riboswitch, or the compound defined above, or theabove-mentioned composition, said method comprising determining whethersaid pathogen bears a guanine riboswitch that controls the expression ofguaA.

In a specific embodiment of the method, the method is method ofselecting a pathogen treatable by (i) the compound defined above; (ii)4-hydroxy-2,5,6-triaminopyrimidine (formula 1.01); (iii)4,5-diamino-6-hydroxy-2-mercaptopyrimidine (formula 1.16); (iv)2,4-diamino-6-hydroxypyrimidine (formula 1.13); (v)5-amino-2-chloro-2,3-dihydrothiazolo[4,5]pyrimidine-7-(6H)-one (2.17);or (vi) the above-mentioned composition, said method comprisingdetermining whether said pathogen bears a guanine riboswitch thatcontrols the expression of guaA.

In accordance with another aspect of the present invention, there isprovided a method of identifying a compound for treating or preventing amicrobial infection caused by a pathogen bearing a guanine riboswitchthat controls the expression of guaA, said method comprising contactingsaid pathogen with a candidate compound and determining the effect ofsaid compound of the growth or survival of said pathogen, wherein adecrease in the growth or survival of said pathogen in the presence ascompared to in the absence of said candidate compound is an indicationthat said compound is suitable for treating or preventing said microbialinfection.

In accordance with another aspect of the present invention, there isprovided a method of identifying a compound for preventing or treating amicrobial infection caused by a pathogen bearing a guanine riboswitchthat controls the expression of guaA, said method comprising contactinga guanine riboswitch with said compound; determining whether saidcompound binds to said guanine riboswitch; wherein the binding of saidcompound to said guanine riboswitch is an indication that said compoundis suitable for treating said microbial infection.

In a specific embodiment of the method, said guanine riboswitch is theguanine xpt riboswitch from Streptococcus pyogenes (STPY-xpt). Inanother specific embodiment, the method further comprises contactingsaid guanine riboswitch with guanine or a guanine-like ligand. Inanother specific embodiment, the method further comprises determiningwhether said compound may be ribosylated.

In accordance with another aspect of the present invention, there isprovided a method for preventing the development of multi-drugresistance of a bacteria in a subject, said method comprisingadministering a non ribosylable ligand of a guanine riboswitch to thesubject, wherein the bacteria bears a guanine riboswitch that controlsthe expression of guaA.

In accordance with another aspect of the present invention, there isprovided a method for preventing the development of multi-drugresistance of a bacteria in a subject, or treating a multidrugresistance of a bacteria in the subject said method comprisingadministering a non ribosylable ligand of a guanine riboswitch to thesubject, wherein the bacteria bears a guanine riboswitch that controlsthe expression of guaA.

In a specific embodiment of the method, said non ribosylable ligand of aguanine riboswitch is: (i) the compound defined above; (ii)4-hydroxy-2,5,6-triaminopyrimidine (formula 1.01); (iii)4,5-diamino-6-hydroxy-2-mercaptopyrimidine (formula 1.16); (iv)2,4-diamino-6-hydroxypyrimidine (formula 1.13); or (v)5-amino-2-chloro-2,3-dihydrothiazolo[4,5]pyrimidine-7-(6H)-one (2.17).

In accordance with another aspect of the present invention, there isprovided a use of a non ribosylable ligand of a guanine riboswitch fortreating a multi-drug resistant bacteria or preventing the developmentof a multi-drug resistant bacteria, wherein the bacteria bears a guanineriboswitch that controls the expression of guaA.

In accordance with another aspect of the present invention, there isprovided a use of a non ribosylable ligand of a guanine riboswitch forthe manufacture of a medicament for treating a multi-drug resistantbacteria or preventing the development of a multi-drug resistantbacteria, wherein the bacteria bears a guanine riboswitch that controlsthe expression of guaA.

In accordance with another aspect of the present invention, there isprovided a use of the compound defined above or the above-mentionedcomposition for treating a multi-drug resistant bacteria or preventingthe development of a multi-drug resistant bacteria, wherein the bacteriabears a guanine riboswitch that controls the expression of guaA.

In a specific embodiment of the uses, said non ribosylable ligand of aguanine riboswitch is: (i) the compound defined above; (ii)4-hydroxy-2,5,6-triaminopyrimidine (formula 1.01); (iii)4,5-diamino-6-hydroxy-2-mercaptopyrimidine (formula 1.16); (iv)2,4-diamino-6-hydroxypyrimidine (formula 1.13); or (v)5-amino-2-chloro-2,3-dihydrothiazolo[4,5]pyrimidine-7-(6H)-one (2.17).

In accordance with another aspect of the present invention there isprovided a non ribosylable ligand of a guanine riboswitch (a) forpreventing or treating a microbial infection in a subject, wherein saidmicrobial infection is caused by a pathogen bearing the guanineriboswitch, and wherein the guanine riboswitch controls the expressionof guaA; or (b) for the disinfection, sterilization and/or antisepsis ofan object from a pathogen bearing a guanine riboswitch that controls theexpression of guaA; or (c) for treating a multi-drug resistant bacteriaor preventing the development of a multi-drug resistant bacteria,wherein the bacteria bears a guanine riboswitch that controls theexpression of guaA.

In a specific embodiment of said ligand for the disinfection,sterilization and/or antisepsis of an object, said object is an animalor milk.

In accordance with another aspect of the present invention, there isprovided (i) the compound defined above; (ii)4-hydroxy-2,5,6-triaminopyrimidine (formula 1.01); (iii)4,5-diamino-6-hydroxy-2-mercaptopyrimidine (formula 1.16); (iv)2,4-diamino-6-hydroxypyrimidine (formula 1.13); or (v)5-amino-2-chloro-2,3-dihydrothiazolo[4,5]pyrimidine-7-(6H)-one (2.17),for (a) preventing or treating a microbial infection in a subject,wherein said microbial infection is caused by a pathogen bearing aguanine riboswitch that controls the expression of guaA; or (b) thedisinfection, sterilization and/or antisepsis of an object from apathogen bearing a guanine riboswitch that controls the expression ofguaA; or (c) treating a multi-drug resistant bacteria or preventing thedevelopment of a multi-drug resistant bacteria, wherein the bacteriabears a guanine riboswitch that controls the expression of guaA.

In a specific embodiment of said composition for the disinfection,sterilization and/or antisepsis of an object, said object is an animalor milk.

In accordance with another aspect of the present invention, there isprovided a composition comprising (i) the compound defined in any one ofclaims 1 to 15; (ii) 4-hydroxy-2,5,6-triaminopyrimidine (formula 1.01);(iii) 4,5-diamino-6-hydroxy-2-mercaptopyrimidine (formula 1.16); (iv)2,4-diamino-6-hydroxypyrimidine (formula 1.13); or (v)5-amino-2-chloro-2,3-dihydrothiazolo[4,5]pyrimidine-7-(6H)-one (2.17);and (a) an antibiotic; (b) an antiseptic; (c) a disinfectant; (d) adiluent; (e) an excipient; (f) a pharmaceutically acceptable carrier; or(g) any combination of (a)-(f), said composition being: (aa) forpreventing or treating a microbial infection in a subject, wherein saidmicrobial infection is caused by a pathogen bearing the guanineriboswitch, and wherein the guanine riboswitch controls the expressionof guaA; or (bb) for the disinfection, sterilization and/or antisepsisof an object from a pathogen bearing a guanine riboswitch that controlsthe expression of guaA; or (cc) for treating a multi-drug resistantbacteria or preventing the development of a multi-drug resistantbacteria, wherein the bacteria bears a guanine riboswitch that controlsthe expression of guaA.

In a specific embodiment of the ligands, compounds and compositions foruses described above, said subject is an animal (e.g., cattle such ascow; goat, ewe, ass, horse, pig; cat; dog; etc.). In another specificembodiment, said subject is a cow. In another specific embodiment, saidsubject is a human. In another specific embodiment, said pathogen is abacteria belonging to the genus Staphylococcus or Clostridium. Inanother specific embodiment, said bacteria is Staphylococcus aureus,methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis,Staphylococcus haemolyticus, Clostridium botulinum or Clostridiumdifficile. In another specific embodiment, said infection is a mammarygland infection.

In accordance with another aspect of the present invention, there isprovided a kit comprising the compound defined above or theabove-mentioned composition, and instructions to use same in theprevention or treatment of a microbial infection.

In a specific embodiment of the kit, the kit comprises: (i) the compounddefined above; (ii) 4-hydroxy-2,5,6-triaminopyrimidine (formula 1.01);(iii) 4,5-diamino-6-hydroxy-2-mercaptopyrimidine (formula 1.16); (iv)2,4-diamino-6-hydroxypyrimidine (formula 1.13); (v)5-amino-2-chloro-2,3-dihydrothiazolo[4,5]pyrimidine-7-(6H)-one (2.17);or (vi) the composition defined above, and instructions to use same inthe prevention or treatment of a microbial infection.

In accordance with another aspect of the present invention, there isprovided a method for preparing the compound of formula 2.02

said method comprising (a) reacting diethylacetamido malonate withguanidine hydrochloride in the presence of sodium methoxide and methanolto obtain a solid; (b) dissolving the solid of (a) in an aqueoussolution; (c) subjecting the solution of (b) to an acidic precipitationto obtain a 5-formamino-2-amino-4,6-dihydroxypyrimidine precipitate; (d)dissolving the 5-formamino-2-amino-4,6-dihydroxypyrimidine precipitatein an acid; and (e) precipitating the solution of (d) to obtain thecompound of formula 2.02.

In a specific embodiment of the method, said reacting is performed underreflux. In another specific embodiment, said acidic precipitation of (b)is performed using a hydrochloric acid (HCl) solution. In anotherspecific embodiment, said HCl solution is a 50% HCl solution. In anotherspecific embodiment, dissolving of (d) is performed using sulfuric acid.In another specific embodiment, said sulfuric acid is 12 M sulfuricacid. In another specific embodiment, the method further compriseswashing said solid of (a) with methanol and chloroform. In anotherspecific embodiment, said aqueous solution is water. In another specificembodiment, said precipitating of (e) is performed using tetrahydrofuran(THF).

Other objects, advantages and features of the present invention willbecome more apparent upon reading of the following non-restrictivedescription of specific embodiments thereof, given by way of exampleonly with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

In the appended drawings:

FIG. 1 is a Venn diagram showing the number of genes (including guaA)that are commonly or differentially expressed by a prototypical mastitisS. aureus isolate (SHY97-3906) and S. aureus isolates causing persistentand chronic bovine mastitis (isolates #3, #557 and #1290).

FIG. 2 displays a quantitative PCR analyses showing the relative levelof guaA mRNA expression by mastitis isolates of S. aureus in the in vivocow-collected samples compared to when the corresponding S. aureusisolates were grown in vitro in freshly collected milk from a healthycow. All results were normalized to the level of expression of the S.aureus gene gyrB. The horizontal bar represents the median. RNA samplesfrom S. aureus grown in two different animals were analyzed: cow #5325at day 10 of infection with strain SHY97-3906 (∘), #3 (Δ), #557 (⋄),#1290 (□). The same shapes were used for the four different strains forthe samples coming from cow #307 at day 8 of infection (black) and cow#307 at day 14 (grey). Quantitative PCR was also done in triplicate forthe RNA sample recovered from strain #1290 in cow 5325 at day 10 ofinfection and the error bar is shown accordingly.

FIG. 3A shows a schematic representation of the structure of the operonxpt/pbuX/guaB/guaA from S. aureus as redefined herein with the number ofbase pairs (bps) present in the intergenic regions (numbers on top) andshows the fragment length in base pairs for the regions amplified byRT-PCR in FIG. 3B (numbers underneath the bars 1, 2 and 3). FIG. 3Bshows the results of an RT-PCR analysis of the intergenic regionsbetween xpt and pbuX (1); pbuX and guaB (2); and guaB and guaA (3).

FIG. 4A shows the structure of some guanine riboswitch ligands such asthe compounds: 4-hydroxy-2,5,6-triaminopyrimidine shown as2,5,6-triaminopyrimidine-4-one (compound 1.01),2,4-diamino-6-hydroxypyrimidine shown as 2,6-diaminopyrimidine-4-one(compound 1.13) and 5,6-diamino-uracil (2AU) as well as their predictedhydrogen bonds. Black and grey arrows stand for hydrogen acceptor anddonor groups, respectively. FIG. 4B shows a histogram representing therelative binding affinity of several compounds at two concentrations (1or 10 μM) for a guanine xpt riboswitch from Streptococcus pyogenes(STPY-xpt) as measured using the assay described in Mulhbacher andLafontaine, 2007. Compounds tested include: guanine (G or Gua), adenine(A), xanthine (X), hypoxanthine (H), compound 1.13, compound 1.01,5,6-diamino-uracil (2AU) and 6-thioguanine (6-thio).

FIGS. 5A and 5B show the effect of compound 1.01 on the growth ofvarious bacterial species in which the guanine riboswitch controls (FIG.5B) or not (FIG. 5A) the expression of the guaA gene. While strainsinsensitive to compound 1.01 do not have the expression of guaA undercontrol of a riboswitch (Bs: Bacillus subtilis, Ef: Enterococcusfeacium, Lm: Listeria monocytogenes, STRd: Streptococcus dysgalactiae,STRpy: Streptococus pyogenes, and STRu: Streptococcus uberis), thestrains susceptible to compound 1.01 control guaA expression via ariboswitch mechanism (Bh: Bacillus halodurans, STAh: S. haemolyticus,STAa: S. aureus ATCC29213, SA228a: S. aureus resistant to beta-lactams,erythromycin, ciprofloxacin, gentamicin and tetracycline, MRSAcoI:methicilin resistant S. aureus COL, Cb: Clostridium botulinum; Cd6:virulent Clostridium difficile isolated in Quebec; Cd630: Clostridiumdifficile strain 630 with complete sequenced genome; STAe: S.epidermidis).

FIG. 6A shows the ribosylation reaction leading to the formation ofnucleosides as well as examples of compounds of the invention that wouldprevent this ribosylation to occur. Asterisks (*) denote the positionwhere chemical modification precludes ribosylation. FIG. 6B showstypical antibiograms performed on strains of E. coli ATCC 35695 (lackinga guaA riboswitch) and Methicilin Resistant S. aureus (containing a guaAriboswitch) grown in the absence (1) or presence of 6-thioguanine at aconcentration of 0.5 mg/mL (2) or 1 mg/mL (3). FIG. 6C shows that6-thioguanine is able to be ribosylated and is incorporated into DNA andRNA (Swann et al., 1996), which likely causes itsriboswitch-independent, non-specific antibiotic activity toward both E.coli and S. aureus (as shown in FIG. 6B).

FIGS. 7A-D show the effect of various antibiotic compounds, includingcompounds 1.13 and 1.01, on the growth of S. aureus in vitro. FIG. 7Ashows the growth of S. aureus strain ATCC 29213 as a function of ligandconcentration in the presence of compound 1.13 () or 1.01 (□). FIG. 7Band 7C show the growth of S. aureus strain ATCC 29213 as a function oftime in the absence () or presence of various compounds includingerythromycin (▴), vancomycin (▪), ciprofloxacin (♦), and compound 1.01(▾), at concentrations equivalent to their corresponding minimalinhibitory concentration (MIC) (FIG. 7B) or ¼ of their MIC (. 7C). FIG.7D shows the growth of S. aureus strain ATCC 29213 as a function of timein media alone (), in the presence of compound 1.01 (▾), or in thepresence of compound 1.01 supplemented with GMP (i.e. a moleculenormally synthesized by the enzyme GMP synthetase encoded by guaA) (▴).

FIG. 8A shows the relative gene expression of several S. aureus genesunder control of the guanine riboswitch (xpt, pbuX, guaA and guaB), whenthe bacteria are grown in vitro in the presence of compound 1.01 aloneor in the presence of compound 1.01 supplemented with GMP. Geneexpression was normalized to the expression level of xpt in the presenceof compound 1.01. Genes gyrA and gyrB represent control genes encodinggyrase protein subunits whose expression is not down-regulated in thepresence of GMP. Results represent the averages of three experiments anderror bars represent standard deviations. FIG. 8B shows a schematicrepresentation of the inhibitory effect of compound 1.01 in S. aureus. Aunique guanine riboswitch performs gene expression regulation of allfour guanine-related genes (xpt, pbux, guaA and guaB).Riboswitch-regulated genes appear in grey and the compound 1.01inhibitory effect is indicated by grey bars. A thick bar indicates theeffect of compound 1.01 on expression of guaA. Broken arrows representmultiple synthesis steps that are not shown in the figure. Black ovalsrepresent genes encoding various metabolite transporters.

FIG. 9 presents the colony forming unit (CFU) count obtained from micemammary gland homogenate previously infected with S. aureus. Micemammary glands were treated 4 hours after infection with PBS with orwithout compound 1.01 at 10, 50 or 100 μg/gland. An asterisk indicatesthat the observed differences are statistically significant.

FIG. 10 shows the inability of S. aureus to develop resistance towardcompound 1.01. The MIC of compound 1.01 on the growth of S. aureusstrain ATCC 29213 was determined every 5 passages (up to 30 passages).For comparison, parallel results were obtained with two knownantibiotics, ciprofloxacin and rifampicin.

FIG. 11 shows examples of representative structures of compounds of theinvention derived from formulas 1.0, 2.0 and 3.0. All examples arelacking the required chemistry to undergo ribosylation by bacterial ormammalian enzymes which could lead to broad and non-specific toxicity.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The gene guaA is essential for the survival or virulence of variousmicrobial pathogens and encodes the enzyme GMP synthetase, whichcatalyses the synthesis of GMP (guanosine-5′-monophosphate) (E.C.6.3.5.2) from XMP (xanthosine 5′-monophosphate). The present inventionshowed that, in some microbial pathogens, expression of the essentialgene guaA is under control of the guanine riboswitch. As shown in theaccompanying examples, the guanine riboswitch in S. aureus was foundherein to control the expression of four genes present on the same mRNAtranscript: xpt, pbuX, guaB and guaA. A bioinformatic analysis revealedthat such guanine riboswitches are found in pathogen microorganisms suchas Staphylococcus aureus and Clostridium difficile but not inmicroorganisms such as Bacillus subtilis and Streptococci in which aguanine riboswitch does not control the expression of guaA. See Table 3below for examples of microorganisms which contain or not a guanineriboswitch that controls the expression of guaA.

The present invention also relates to the unexpected discovery that somepathogen microorganisms, such as S. aureus, highly express guaA inspecific environments during infection of an animal host such as amammal. As shown in the accompanying examples, microarray andquantitative PCR gene expression analysis was performed on various S.aureus strains isolated from bovine intramammary infections. Thesestudies reveal that guaA is highly expressed during infection in cows byprototypical S. aureus strains known to cause bovine mastitis as well asin strains known to cause persistent and chronic mastitis. Therefore,the present invention also relates to the surprising discovery thatguanine riboswitches controlling the expression of the guaA gene aresuitable targets for therapeutic antimicrobial intervention.

The present invention also relates to the surprising discovery thatguanine-like compounds modified to be unable to undergo ribosylation,are nevertheless capable of binding to or interacting with riboswitchaptamers and can affect expression of genes that are under riboswitchcontrol. As shown in the accompanying examples, binding and competitionassays measuring the ability of various compounds to displace a knownguanine riboswitch ligand showed that the compounds of the presentinvention are capable of binding to the guanine riboswitch. Microarraygene expression analysis confirmed that the compounds of the presentinvention can not only bind to guanine riboswitches but also modulateexpression of genes under control of the guanine riboswitch.Furthermore, the specificity and mechanism of this modulation wasvalidated by studies combining a compound of the present invention withthe metabolite GMP.

The present invention also relates to the discovery that the compoundsof the present invention can act as specific or selective antimicrobialagents to which microbial pathogens (that have the guaA gene undercontrol of the guanine riboswitch) cannot develop resistance. Thepresent invention also relates to the discovery that the compounds ofthe present invention have no substantial inhibitory activity againstmicroorganisms in which the gene guaA is not controlled by a guanineriboswitch. As shown in the accompanying examples, the compounds of thepresent invention can demonstrate an anti-bacterial effect on S. aureusand C. difficile strains when grown in vitro (e.g., in culture media ormilk) or in vivo (e.g., in mice mammary gland infections). Thespecificity of the compounds of the present invention was demonstratedby their failure to exert an antibacterial effect on strains (e.g., E.coli) lacking a guaA under control of the guanine riboswitch. Theanti-bacterial effect of the compounds of the present invention wereshown herein to be comparable to several well-known antibioticcompounds. Strikingly, unlike the well-known antibiotic compounds,ciprofloxacin and rifampicin, a S. aureus lstrain did not developantibiotic resistance to a compound of the present invention. Therefore,it is also disclosed herein that the compounds of the present inventionare able to inhibit the expression of the gene guaA, preventing rapiddevelopment of bacterial resistance in S. aureus.

The present invention also relates to compounds of the following generalformulas:

Guanine

Formula 1.0:

Formula 1.01 (4-hydroxy-2,5,6-triaminopyrimidine here shown as theguanine riboswitch ligand 2,5,6-triaminopyrimidine-4-one):

Formula 1.13 (2,4-diamino-6-hydroxypyrimidine here shown as the guanineriboswitch ligand 2,6-diaminopyrimidine-4-one):

Formula 1.16 (4,5-diamino-6-hydroxy-2-mercaptopyrimidine here shown asthe guanine riboswitch ligand 5,6-diamino-2-mercaptopyrimidine-4-one)

Formula 2.0:

Formula 2.02 (9-oxoguanine here shown as5-Amino-6H-oxazolo[5,4-d]pyrimidin-7-one)

Formula 2.17(5-amino-2-chloro-2,3-dihydrothiazolo[4,5]pyrimidine-7-(6H)-one)

Formula 3.0:

The compounds of the present invention bind to the guanine riboswitchbinding site and yet are chemically unable to be ribosylated by thetargeted pathogens. This property prevents incorporation of thecompounds into cellular nucleosides, nucleotides or nucleic acids, andthus prevents broad, non-specific toxicity for bacterial species nottargeted by the compound and for the mammalian or animal host in whichthe present compounds are used for therapeutic intervention.

Definitions

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one” butit is also consistent with the meaning of “one or more”, “at least one”,and “one or more than one”.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, un-recitedelements or method steps.

As used herein, the terms “molecule”, “compound”, “agent” or “ligand”are used interchangeably and broadly to refer to natural, synthetic orsemi-synthetic molecules or compounds. The term “compound” thereforedenotes, for example, chemicals, macromolecules, cell or tissue extracts(from plants or animals) and the like. Non-limiting examples ofcompounds include peptides, antibodies, carbohydrates, nucleic acidmolecules and pharmaceutical agents. The compound can be selected andscreened by a variety of means including random screening, rationalselection and by rational design using, for example, ligand modelingmethods such as computer modeling. The terms “rationally selected” or“rationally designed” are meant to define compounds which have beenchosen based on the configuration of interacting domains of the presentinvention (e.g., the guanine riboswitch). As will be understood by theperson of ordinary skill, molecules having non-naturally occurringmodifications are also within the scope of the term “compound”. Forexample, the compounds of the present invention can be modified toenhance their activity, stability, toxicity and/or bioavailability. Thecompounds or molecules identified in accordance with the teachings ofthe present invention have a therapeutic value in diseases or conditionsrelated to microbial infections.

The term “subject” or “patient” as used herein refers to an animal,preferably a mammal such as but not limited cattle such as cow; goat;ewe; ass; horse; pig; cat; dog; etc. or human who is the object oftreatment, observation or experiment.

As used herein, the terms “pharmaceutically acceptable” refer tomolecular entities and compositions that are physiologically tolerableand do not typically produce an allergic or similar untoward reaction,such as gastric upset, dizziness and the like, when administered toanimals (e.g., cows) or human. Preferably, as used herein, the term“pharmaceutically acceptable” means approved by regulatory agency of thefederal or state government or listed in the U.S. Pharmacopeia or othergenerally recognized pharmacopeia for use in animals, and moreparticularly in humans.

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which the compounds of the present invention may be administered.Sterile water or aqueous saline solutions and aqueous dextrose andglycerol solutions may be employed as carriers, particularly forinjectable solutions. Suitable pharmaceutical carriers are described in“Remington's Pharmaceutical Sciences” by E. W. Martin.

Compounds of the invention may be administered in a pharmaceuticalcomposition. Pharmaceutical compositions may be administered in unitdosage form. Any appropriate route of administration may be employed,for example, parenteral, subcutaneous, intramuscular, intramammary,intracranial, intraorbital, ophthalmic, intraventricular, intracapsular,intraarticular, intraspinal, intracisternal, intraperitoneal,intranasal, aerosol, or oral administration. Examples of specific routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, intramammary; oral (e.g., inhalation); transdermal(topical); transmucosal, and rectal administration.

Conventional pharmaceutical practice may be employed to provide suitableformulations or compositions to administer such compositions topatients. Methods well known in the art for making pharmaceuticalcompositions and formulations are found in, for example, Remington: TheScience and Practice of Pharmacy, (20th ed.) ed. A. R. Gennaro A R.,2000, Lippincott: Philadelphia.

Therapeutic formulations for oral administration, may be in the form oftablets or capsules; ; for transmucosal (e.g., rectal, intranasal) ortransdermal/percutaneous administration may be in the form of ointments,powders, nasal drops, sprays/aerosols or suppositories; for topicaladministration, may be in the form of ointments, creams, gels orsolutions; for parenteral administration (e.g., intravenously,intramuscularly, intradermal, intramammary, subcutaneously,intrathecally or transdermally), using for example injectable solutions.Furthermore, administration can be carried out sublingually or asophthalmological preparations or as an aerosol, for example in the formof a spray. Intravenous, intramuscular or oral administration is apreferred form of use.

Oral

For the purpose of oral therapeutic administration, the active compoundcan be incorporated with excipients and used for example in the form oftablets, troches, dragees, hard or soft gelatin capsules, solutions(e.g., syrups), emulsions or suspensions, or capsules. For thepreparation of formulations for oral administration, the compounds ofthe present invention may be admixed with pharmaceutically inert,inorganic or organic excipients (e.g., pharmaceutically compatiblebinding agents, and/or adjuvant). The tablets, pills, capsules, trochesand the like can contain any of the following ingredients, or compoundsof a similar nature: a binder such as microcrystalline cellulose, gumtragacanth or gelatin; an excipient such as starch or lactose, adisintegrating agent such as alginic acid, Primogel, or corn starch; alubricant such as magnesium stearate or Sterotes; a glidant such ascolloidal silicon dioxide; a sweetening agent such as sucrose orsaccharin; or a flavoring agent such as peppermint, methyl salicylate,or orange flavoring. Examples of suitable excipients for tablets,dragees or hard gelatin capsules for example include lactose, maizestarch or derivatives thereof, talc or stearic acid or salts thereof.Suitable excipients for use with soft gelatin capsules include forexample vegetable oils, waxes, fats, semi-solid or liquid polyols etc.;according to the nature of the active ingredients it may however be thecase that no excipient is needed at all for soft gelatin capsules.

For the preparation of solutions and syrups, excipients which may beused include for example water, polyols, saccharose, invert sugar andglucose.

Nasal

For administration by inhalation, the compounds may be delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer. Formulations for inhalation may contain excipients, orexample, lactose, or may be aqueous solutions containing, for example,polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may beoily solutions for administration in the form of nasal drops, or as agel.

Transmucosal or Transdermal

For transmucosal or transdermal administration, penetrants appropriateto the barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art, and include, for example, fortransmucosal administration, detergents, bile salts, and fusidic acidderivatives. Transmucosal administration can be accomplished through theuse of nasal sprays or suppositories. For transdermal administration,the active compounds are formulated into ointments, salves, gels, orcreams as generally known in the art. For suppositories, and local orpercutaneous application, excipients which may be used include forexample natural or hardened oils, waxes, fats and semi-solid or liquidpolyols.

Parenteral

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. Solutions or suspensions used for parenteralapplication can include the following components: a sterile diluent suchas water for injection (where water soluble), saline solution, fixedoils (e.g., paraffin oil), polyalkylene glycols such as polyethyleneglycols, glycerine, propylene glycol or other synthetic solvents, oilsof vegetable origin, or hydrogenated napthalenes; antibacterial agentssuch as benzyl alcohol or methyl parabens; antioxidants such as ascorbicacid or sodium bisulfite; chelating agents such asethylenediaminetetraacetic acid; reducing agents such as dithiothreitol,buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. The pH canbe adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. Biocompatible, biodegradable lactide polymer,lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylenecopolymers may be used to control the release of the compounds. Otherpotentially useful parenteral delivery systems for compounds of theinvention include ethylenevinyl acetate copolymer particles, osmoticpumps, implantable infusion systems, and liposomes. The parenteralpreparation can also be enclosed in ampoules, disposable syringes ormultiple dose vials made of glass or plastic.

For intravenous or intramammary administration, suitable carriersinclude physiological saline, bacteriostatic water, Cremophor EL™ (BASF,Parsippany, N.J.) or phosphate buffered saline (PBS).

Liposomal suspensions (including liposomes targeted to specific celltypes) can also be used as pharmaceutically acceptable carriers. Avariety of liposomal formulations suitable for delivering a compound toan animal have been described and demonstrated to be effective indelivering a variety of compound, including, e.g., small molecules,nucleic acids, and polypeptides.

As mentioned earlier, medicaments containing the compounds of thepresent invention are also an object of the present invention, as is aprocess for the manufacture of such medicaments, which process comprisesbringing one or more of the compounds of the present invention to, ifdesired, one or more other therapeutically valuable substances into agalenical administration form.

Salts, Esters, Hydrates and Solvates

The compounds of the present invention include pharmacologicallyacceptable salts and ester derivatives thereof as well as hydrates orsolvates thereof and all stereoisomeric forms of the referencedcompounds. The compounds and pharmacologically acceptable esters thereofof the present invention can form pharmacologically acceptable salts ifnecessary.

Salts

The terms “pharmacologically acceptable salt thereof” refer to a salt towhich the compounds of the present invention can be converted. Preferredexamples of such a salt include alkali metal salts such as a sodiumsalt, a potassium salt, a lithium salt, magnesium or calcium salts;alkaline earth metal salts such as a calcium salt and a magnesium salt;metal salts such as an aluminium salt, an iron salt, a zinc salt, acopper salt, a nickel salt and a cobalt salt; amine salts such asinorganic salts including an ammonium salt; organic salts or ammoniumsaltssuch as a t-octylamine salt, a dibenzylamine salt, a morpholinesalt, a glucosamine salt, a phenylglycine alkyl ester salt, anethylenediamine salt, an N-methylglucamine salt, a guanidine salt, adiethylamine salt, a triethylamine salt, a dicyclohexylamine salt, anN,N′-dibenzylethylenediamine salt, a chloroprocaine salt, a procainesalt, a diethanolamine salt, an N-benzyl-phenethylamine salt, apiperazine salt, a tetramethylammonium salt and atris(hydroxymethyl)aminomethane salt; inorganic acid salts such ashydrohalic acid salts such as a hydrofluoride, a hydrochloride, ahydrobromide or a hydroiodide, a nitrate, a perchlorate, a sulfate or aphosphate; lower alkanesulfonates such as a methanesulfonate,trifluoromethanesulfonate or an ethanesulfonate; arylsulfonates such asa benzenesulfonate or a p-toluenesulfonate and the like, which are nontoxic to living organisms; organic acid salts such as an acetate, amalate, adipate, a fumarate, a succinate, a citrate, alginate,ascorbate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate,camphorsulfonate, cinnamate, cyclopentanepropionate, digluconate,dodecylsulfate, ethanesulfonate, glucoheptanoate, glycerophosphate,hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,hydroiodide, 2- hydroxyethanesulfonate, itaconate, lactate, maleate,mandelate, sulfonate, methanesulfonate, trifluoromethanesulfonates,ethanesulfonates 2-naphthalenesulfonate, nicotinate, nitrate, oxalate,pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate,propionate, tartrate, thiocyanate, tosylate, and undecanoate, atartrate, an oxalate or a maleate; and amino acid salts such as aglycine salt, a lysine salt, an arginine salt, an omithine salt,histidine, a glutamate or an aspartate salt. Additionally, basicnitrogen containing groups may be quaternized with such agents as loweralkyl halides such as methyl, ethyl, propyl, and butyl chlorides,bromides and iodides; dialkyl sulfates including dimethyl, diethyl, anddibutyl sulfate; and diamyl sulfates, long chain halides such as decyl,lauryl, myristyl and strearyl chlorides, bromides and iodides, aralkylhalides including benzyl and phenethyl bromides, and others. For furtherexample, see S. M. Berge, et al. “Pharmaceutical Salts,” J. Pharm. Sci.1977, 66, 1-19. Such salts can be formed quite readily by those skilledin the art using standard techniques.

Preferred example of the salts formed with an acidic group present inthe compounds of the present invention include metal salts such asalkali metal salts (e.g., sodium salts, potassium salts and lithiumsalts), alkali earth metal salts (e.g., calcium salts and magnesiumsalts), aluminum salts and iron salts; amine salts such as inorganicamine salts (e.g., ammonium salts) and organic amine salts (e.g.,t-octylamine salts, dibenzylamine salts, morpholine salts, glucosaminesalts, phenylglycinealkyl ester salts, ethylenediamine salts,N-methylglucamine salts, guanidine salts, diethylamine salts,triethylamine salts, dicyclohexylamine salts,N,N′-dibenzylethylenediamine salts, chloroprocaine salts, procainesalts, diethanolamine salts. N-benzylphenethylamine salts, piperazinesalts, tetramethylammonium salts and tris(hydroxymethyl)aminomethanesalts; and amino acid salts such as glycine salts, lysine salts,arginine salts, ornithine salts, glutamates and aspartates.

All salts are intended to be pharmaceutically acceptable salts withinthe scope of the invention and all salts are considered equivalent tothe free forms of the corresponding compounds for purposes of theinvention.

Esters

Physiologically/pharmaceutically acceptable esters are also useful asactive medicaments. The term “pharmaceutically acceptable esters”embraces esters of the compounds of the present invention, in whichhydroxy groups (e.g., in carboxylic acid) have been converted to thecorresponding esters and may act as a prodrug which, when absorbed intothe bloodstream of a warm-blooded animal, may cleave in such a manner asto release the drug form and permit the drug to afford improvedtherapeutic efficacy. Such esters can be formed with inorganic ororganic acids such as nitric acid, sulphuric acid, phosphoric acid,citric acid, formic acid, maleic acid, acetic acid, succinic acid,tartaric acid, methanesulphonic acid, p-toluenesulphonic acid and thelike, which are non toxic to living organisms. Further examples are theesters with aliphatic or aromatic acids such as acetic acid or withaliphatic alcohol (e.g., alkyl esters, including methyl, ethyl, propyl,isopropyl, butyl, isobutyl or pentyl esters, and the like) or aromaticalcohols (e.g., benzyl ester)

Esters can be prepared from their corresponding acids or salts by avariety of methods known to those skilled in the art, such as, forexample, by first transforming the acid to the acid chloride and thenreacting the acid chloride with a suitable alcohol. Other suitablemethods for making esters are described in Kemp and Vellaccio, 1980.

Where esters of the invention have a basic group, such as an aminogroup, the compound can be converted to a salt by reacting it with anacid, and in the case where the esters have an acidic group, such as asulfonamide group, the compound can be converted to a salt by reactingit with a base. The compounds of the present invention encompass suchsalts.

Salts and esters of the compounds of the present invention may beprepared by known method by employing appropriate starting materials orintermediate compounds that are readily available and/or are describedherein.

Generally, a desired salt of a compound of this invention can beprepared in situ during the final isolation and purification of acompound by means well known in the art. For example, a desired salt canbe prepared by separately reacting the purified compound in its freebase or free acid form with a suitable organic or inorganic acid, orsuitable organic or inorganic base, respectively, and isolating the saltthus formed. In the case of basic compounds, for example, the free baseis treated with anhydrous HCl in a suitable solvent such as THF, and thesalt isolated as a hydrochloride salt. In the case of acidic compounds,the salts may be obtained, for example, by treatment of the free acidwith anhydrous ammonia in a suitable solvent such as ether andsubsequent isolation of the ammonium salt. These methods areconventional and would be readily apparent to one skilled in the art.

The compounds of this invention may be esterified by a variety ofconventional procedures including reacting the appropriate anhydride,carboxylic acid or acid chloride with the alcohol group of a compound ofthis invention. The appropriate anhydride is reacted with the alcohol inthe presence of a base to facilitate acylation such as1,8-bis[dimethylamino]naphthalene or N,N-dimethylaminopyridine. Or, anappropriate carboxylic acid can be reacted with the alcohol in thepresence of a dehydrating agent such as dicyclohexylcarbodiimide,1-[3-dimethylaminopropyl]-3-ethylcarbodiimide or other water solubledehydrating agents which are used to drive the reaction by the removalof water, and, optionally, an acylation catalyst. Esterification canalso be effected using the appropriate carboxylic acid in the presenceof trifluoroacetic anhydride and, optionally, pyridine, or in thepresence of N,N-carbonyldiimidazole with pyridine. Reaction of an acidchloride with the alcohol can be carried out with an acylation catalystsuch as 4-DMAP or pyridine.

One skilled in the art would readily know how to successfully carry outthese as well as other known methods of esterification of alcohols.

Hydrates

As used herein the terms, “pharmaceutically acceptable hydrate” refer tothe compounds of the instant invention crystallized with one or moremolecules of water to form a hydrated form.

Prodrugs and Solvates

Prodrugs and solvates of the compounds of the invention are alsocontemplated herein. A discussion of prodrugs is provided in T. Higuchiand V. Stella, Pro-drugs as Novel Delivery Systems (1987) 14 of theA.C.S. Symposium Series, and in Bioreversible Carriers in Drug Design,(1987) Edward B. Roche, ed., American Pharmaceutical Association andPergamon Press. The term “prodrug” means a compound (e.g., a drugprecursor) that is transformed in vivo to yield a compound of thepresent invention or a pharmaceutically acceptable salt, hydrate orsolvate of the compound. The transformation may occur by variousmechanisms (e.g., by metabolic or chemical processes), such as, forexample, through hydrolysis in blood. A discussion of the use ofprodrugs is provided by T. Higuchi and W. Stella, “Pro-drugs as NovelDelivery Systems,” Vol. 14 of the A.C.S. Symposium Series, and inBioreversible Carriers in Drug Design, ed. Edward B. Roche, AmericanPharmaceutical Association and Pergamon Press, 1987.

For example, if a compound of the present invention or apharmaceutically acceptable salt, hydrate or solvate of the compoundcontains a carboxylic acid functional group, a prodrug can comprise anester formed by the replacement of the hydrogen atom of the acid groupwith a group such as, for example, (C1-C8)alkyl,(C2-C12)alkanoyloxymethyl, 1-(alkanoyloxy)ethyl having from 4 to 9carbon atoms, 1-methyl-1-(alkanoyloxy)-ethyl having from 5 to 10 carbonatoms, alkoxycarbonyloxymethyl having from 3 to 6 carbon atoms,1-(alkoxycarbonyloxy)ethyl having from 4 to 7 carbon atoms,1-methyl-1-(alkoxycarbonyloxy)ethyl having from 5 to 8 carbon atoms,N-(alkoxycarbonyl)aminomethyl having from 3 to 9 carbon atoms,1-(N-(alkoxycarbonyl)amino)ethyl having from 4 to 10 carbon atoms,3-phthalidyl, 4-crotonolactonyl, gamma-butyrolacton-4-yl,di-N,N—(C1-C2)alkylamino(C2-C3)alkyl (such as δ-dimethylaminoethyl),carbamoyl-(C1-C2)alkyl, N,N-di(C1-C2)alkylcarbamoyl-(C1-C2)alkyl andpiperidino-, pyrrolidino- or morpholino(C2-C3)alkyl, and the like.

Similarly, if a compound of the present invention contains an alcoholfunctional group, a prodrug can be formed by the replacement of thehydrogen atom of the alcohol group with a group such as, for example,(C1-C6)alkanoyloxymethyl, 1-((C1-C6)alkanoyloxy)ethyl,1-methyl-1-((C1-C6)alkanoyloxy)ethyl, (C1-C6)alkoxycarbonyloxymethyl,N—(C1-C6)alkoxycarbonylaminomethyl, succinoyl, (C1-C6)alkanoyl,α-amino(C1-C4)alkanyl, arylacyl and α-aminoacyl, orα-aminoacyl-α-aminoacyl, where each α-aminoacyl group is independentlyselected from the naturally occurring L-amino acids, P(O)(OH)2,—P(O)(O(C1C6)alkyl)2 or glycosyl (the radical resulting from the removalof a hydroxyl group of the hemiacetal form of a carbohydrate), and thelike.

If a compound of the present invention incorporates an amine functionalgroup, a prodrug can be formed by the replacement of a hydrogen atom inthe amine group with a group such as, for example, R-carbonyl,RO-carbonyl, NRR′-carbonyl where R and R′ are each independently(C1-C10)alkyl, (C3-C7)cycloalkyl, benzyl, or R-carbonyl is a naturalα-aminoacyl or natural α-aminoacyl, —C(OH)C(O)O Y1 wherein Y1 is H,(C1-C6)alkyl or benzyl, —C(OY2)Y3 wherein Y2 is (C1-C4) alkyl and Y3 is(C1-C6)alkyl, carboxy (C1-C6)alkyl, amino(C1-C4)alkyl or mono-N— ordi-N,N—(C1-C6)alkylaminoalkyl, —C(Y4)Y5 wherein Y4 is H or methyl and Y5is mono-N—or di-N,N—(C1-C6)alkylamino morpholino, piperidin-1-yl orpyrrolidin-1-yl, and the like.

One or more compounds of the invention may exist in unsolvated as wellas solvated forms with pharmaceutically acceptable solvents such aswater, ethanol, and the like, and it is intended that the inventionembrace both solvated and unsolvated forms. “Solvate” means a physicalassociation of a compound of this invention with one or more solventmolecules. This physical association involves varying degrees of ionicand covalent bonding, including hydrogen bonding. In certain instancesthe solvate will be capable of isolation, for example when one or moresolvent molecules are incorporated in the crystal lattice of thecrystalline solid. “Solvate” encompasses both solution-phase andisolatable solvates. Non-limiting examples of suitable solvates includeethanolates, methanolates, and the like. “Hydrate” is a solvate whereinthe solvent molecule is H₂O.

One or more compounds of the invention may optionally be converted to asolvate. Preparation of solvates is generally known. Thus, for example,M. Caira et al, J. Pharmaceutical Sci., 93(3), 601-611 (2004) describethe preparation of the solvates of the antifungal fluconazole in ethylacetate as well as from water. Similar preparations of solvates,hemisolvate, hydrates and the like are described by E. C. van Tonder etal, AAPS Pharm Sci Tech., 5(1), article 12 (2004); and A. L. Bingham etal, Chem. Commun., 603-604 (2001). A typical, non-limiting, processinvolves dissolving the inventive compound in desired amounts of thedesired solvent (organic or water or mixtures thereof) at a higher thanambient temperature, and cooling the solution at a rate sufficient toform crystals which are then isolated by standard methods. Analyticaltechniques such as, for example I. R. spectroscopy, show the presence ofthe solvent (or water) in the crystals as a solvate (or hydrate).

Stereoisomers, Enantiomers, Racemates, Tautomers

The compounds of the present invention have asymmetric carbon atoms andcan exist in the form of optically pure enantiomers or as racemates. Theinvention embraces all of these forms.

For purposes of this Specification, “pharmaceutically acceptabletautomer” means any tautomeric form of any compound of the presentinvention.

The purification of enantiomers and the separation of isomeric mixturesof a compound of the present invention may be accomplished by standardtechniques known in the art.

The pharmaceutical compositions may also contain preserving agents,solubilising agents, stabilising agents, wetting agents, emulsifiers,sweeteners, colorants, odorants, salts for the variation of osmoticpressure, buffers, coating agents or antioxidants. As mentioned earlier,they may also contain other therapeutically valuable agents.

Dosages

The dosages in which the compounds of the present invention areadministered in effective amounts depend on the nature of the specificactive ingredient, the age and the requirements of the patient and themode of application. In general, daily dosages of about 1 mg -1000 mg,preferably 5 mg -500 mg, per day come into consideration.

The skilled artisan will appreciate that certain factors may influencethe dosage required to effectively treat a subject, including but notlimited to the severity of the disease or disorder, previous treatments,the general health and/or age of the subject, and other diseasespresent. Moreover, treatment of a subject with a therapeuticallyeffective amount of a compound of the present invention can include aseries of treatments.

Toxicity and Therapeutic Efficacy

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds that exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

Data obtained from cell culture assays and animal studies can be used informulating a range of dosage for use in humans. The dosage of suchcompounds lies preferably within a range of circulating concentrationsthat include the ED₅₀ with little or no toxicity. The dosage may varywithin this range depending upon the dosage form employed and the routeof administration utilized. For any compound used in the method of theinvention, the therapeutically effective dose can be estimated initiallyfrom cell culture assays. A dose may be formulated in animal models toachieve a circulating plasma concentration range that includes the IC50(i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma may be measured, for example, by highperformance liquid chromatography.

Kits

The present invention also encompasses kits comprising the compounds ofthe present invention. For example, the kit can comprise one or morecompounds or agents binding to a guanine riboswitch and inhibit orreduce the expression of the gene guaA. The kit may optionally includeone or more control samples. The compounds or agents can be packaged ina suitable container. The kit can further comprise instructions forusing the kit.

The present invention also relates to methods for preparing theabove-mentioned compounds. In an embodiment, the above-mentioned methodis that defined in Examples 13 and 16 below.

The present invention is illustrated in further details by the followingnon-limiting examples.

EXAMPLE 1 guaA Expression in S. aureus Isolates from Infected Cows andfrom Milk

The present invention relates to the discovery that some pathogenmicroorganisms, such as S. aureus, sustain a significant level ofexpression of the gene guaA (comparable to some known essential genes)in specific environments during infection of a mammalian or animal host.The present invention also discloses the sustained expression of guaA inseveral S. aureus isolates, including those causing persistent andchronic bovine mastitis, during infection in a cow. The presentinvention also discloses that the expression of guaA in several S.aureus isolates, including those causing persistent and chronic bovinemastitis, was relatively higher during infection in cows compared to theexpression of guaA in the corresponding S. aureus isolates grown invitro in freshly collected milk obtained from a healthy cow.

DNA microarray gene expression analysis was used to identify genes thatwere expressed (at different time points) by various S. aureus strainsin vivo during bovine mastitis. Mastitis infection in two cows wereexamined: Cow #310 and cow #5325. The results of this analysis withrespect to all genes are summarized in the Venn diagram presented inFIG. 1. Briefly, the chip was produced from genes amplified and selectedin inventor's laboratory. FIG. 1 shows the number of genes (includingguaA) that are commonly or differentially expressed by a prototypical S.aureus mastitis isolate (SHY97-3906) and S. aureus isolates causingpersistent and chronic bovine mastitis (isolates #3, #557 and #1290).The results of this microarray analysis with respect to expression ofguaA, are summarized in Table 1 below. Although guaA mRNA expression wasdetected in all samples analyzed, most of the samples showed that guaAwas expressed at a level higher than the average level of expression forall genes detected on the arrays (Table 1). These results appear toindicate that, even though the different S. aureus strains (prototypicaland chronic) show some differences in the genes they each express, guaAis commonly expressed by both the prototypical mastitis strain(SHY97-3906) and the group of chronic strains (#3, #557 and #1290).Also, the level of expression of guaA, measured by real-time PCR forstrain #1290 isolated from three different samples of mastitic milk invivo, was very similar to that measured for gyrB (1.19 ±0.33 fold vs.gyrB), demonstrating that guaA is expressed during infection at levelscomparable to the well-known S. aureus essential gene gyrB, whichencodes an essential DNA gyrase (Anderson et al., 2000).

TABLE 1 Mastitic milk samples in which the expression of guaA was shownto be greater than the average expression of all genes on microarraysfor 4 different S. aureus strains at 2 different time points in twoanimals. S. aureus strains Cow Day of infection SHY97-3609 3 557 1290310 8     310 10     5325 10



Symbols:  , expression of guaA greater than the average expression ofall genes on arrays;

 , expression of guaA was detected but was below the average expressionof all genes on arrays;

 , not tested by DNA arrays.

Depicted in FIG. 2 are quantitative PCR analyses showing the relativelevel of guaA mRNA expression by mastitis isolates of S. aureus in thein vivo cow-collected samples compared to when the corresponding S.aureus isolates were grown in vitro in freshly collected milk from ahealthy cow. All results were normalized using the level of expressionof S. aureus essential gene, gyrB. The horizontal bar represents themedian. RNA samples from S. aureus grown in two different animals wereanalyzed: cow #5325 at day 10 of infection with strain SHY97-3906 (∘),#3 (⋄), #557 (□), #1290 (□). The same shapes were used for the fourdifferent strains for the samples coming from cow #307 at day 8 ofinfection (black) and cow #307 at day 14 (grey). Quantitative PCR wasalso done in triplicate for the RNA sample recovered from strain #1290in cow 5325 at day 10 of infection and the error bar is shownaccordingly.

Briefly, four different Staphylococcus aureus strains (clinical isolateSHY97-3906 and chronic isolates 3, 557 and 1290) were inoculated in 8multiparous Holstein cows in mid lactation at the Dairy and SwineResearch and Development Centre of Agriculture and Agri-Food Canada inSherbrooke, QC, Canada. Each quarter of the mammary gland of the cowswas infused with 50 CFU of each S. aureus strain. After the inoculation,milk was collected from each quarter of each cow every 2-4 days in themorning for a total of 18 days. S. aureus was then isolated from themastitic milk and RNA was extracted from the in vivo grown S. aureus.With the RNA, fluorescent probes for hybridization to DNA arrays weregenerated through an aminoallyl cDNA labelling procedure. Thefluorescent probes (labeled with Cy5) were hybridized to a DNA arraythat contained a selection of 530 known or putative genes. Only geneswith a Cy5 signal intensity of ≧100%, i.e., greater or equal to the meanCy5 intensity of the entire array were analyzed. The results wereconfirmed by quantitative PCR and the level of expression of genes foundto be strongly expressed during mastitis by S. aureus was compared tothat of in vitro grown S. aureus. FIG. 2 shows that guaA is expressed atlevel that is roughly equivalent to or higher than the essential genegyrB in S. aureus present in mastitic milk (Mayer et al, 1988).

EXAMPLE 2 Polycistronic RNA Encoding xpt/pbuX/guaB/guaA

The present invention relates to the discovery that the guanineriboswitch of S. aureus controls the expression of a polycistronic mRNAencoding xpt/pbuX/guaB/guaA and not only the non-essential genes xpt andpbuX, contradicting the teachings of the prior art that the S. aureusguanine riboswitch controls genes that are not essential for survival orvirulence of the pathogen. Example 1 showed that there was a sustainedexpression of guaA in S. aureus during infection in cows.

Shown in FIG. 3A is a schematic representation of the structure of theoperon xpt/pbuX/guaB/guaA from S. aureus as redefined herein with thenumber of base pairs present in the intergenic regions indicated. A grayrectangle represents the guanine riboswitch. Annotated arrows indicatethe number of bases between adjacent genes. This schematicrepresentation was compiled from the results of an RT-PCR analysis oftotal RNA from S. aureus (FIG. 3B) of the intergenic regions between xptand pbuX (1); pbuX and guaB (2); and guaB and guaA (3). Briefly, totalRNA extract from S. aureus was submitted to DNAse1 followed by reversetranscription. The obtained cDNA was then amplified by PCR using thefollowing primer designed to amplify intergenic region. These resultsclearly demonstrate the polycistronic nature of the mRNA transcriptdownstream of the guanine riboswitch.

EXAMPLE 3 Ability of Compounds of the Invention to Bind GuanineRiboswitches

The compounds of the present invention are able to compete withguanine-like ligands for binding to the guanine riboswitch. Structuralmodifications of these and other related compounds influence thisproperty.

Shown in FIG. 4A is the structure of the guanine ligand and thecompounds: 4-hydroxy-2,5,6-triaminopyrimidine shown as2,5,6-triaminopyrimidine-4-one (compound 1.01),2,4-diamino-6-hydroxypyrimidine shown as 2,6-diaminopyrimidine-4-one(compound 1.13) and 5,6-diamino-uracil (2AU) as well as their predictedhydrogen bonds (arrows). FIG. 4B shows a histogram representing therelative binding affinity of several compounds (i.e. guanine (G or Gua),adenine (A), xanthine (X), hypoxanthine (H), compound 1.13, compound1.01, 5,6-diamino-uracil (2AU) and 6-thioguanine (6-thio)) at twoconcentrations (1 or 10 μM) for a guanine xpt riboswitch fromStreptococcus pyogenes (STPY-xpt). The experiment was performed as infor example Mulhbacher and Lafontaine, 2007. Compounds 1.01 and 1.13were purchased from Fluka product numbers 17376 and 33050 respectively.Briefly, STPY-xpt aptamers were incubated in the presence of theguanine-like ligand 2-aminopurine alone (Neg. control) or in thepresence of 1 or 10 μM of the above-listed compounds. The relativebinding affinity was obtained from the ability of a ligand to displacethe 2-aminopurine from the guanine riboswitch STPY-xpt aptamer. Theresults comparing the relative binding of 1 μM guanine with 10 μM of thevarious tested compounds are summarized in Table 2 below. The phrase“appropriate properties” is meant to refer to structural characteristicsenabling compounds to fit or interact with the active site of theguanine riboswitch, while lacking the chemistry required to undergoribosylation, thus precluding the compounds incorporation into cellularnucleosides, nucleotides or nucleic acids of non targeted bacteria or ofthe mammalian host treated with the compounds.

TABLE 2 Description of the properties of various known guanineriboswitch ligands Relative Appropriate Compound Structure bindingRibosylation properties guanine (1 μM)

1 Yes No hypoxanthine (10 μM)

0.50 Yes No xanthine (10 μM)

0.08 Yes No adenine (10 μM)

0.05 Yes No 6-thioguanine (10 μM)

0.14 Yes No 5,6-diamino-uridine (10 μM)

0.05 Yes No Compound 1.01 (10 μM)

0.26 No Yes Compound 1.13 (10 μM)

0.39 No Yes

EXAMPLE 4 Growth Inhibition of Bacterial Species Possessing a GuanineRiboswitch that Controls the Expression of the guaA Gene by Compounds ofthe Present Invention

As disclosed herein, compounds of the present invention (such as4-hydroxy-2,5,6-triaminopyrimidine (compound 1.01)) are able to inhibitthe growth of bacteria if such bacteria possess a guanine riboswitchthat controls the expression of the guaA gene. Also, the compounds ofthe present invention (e.g., compound 1.01) do not substantially inhibitthe growth of bacteria that have the guaA gene but not the guanineriboswitch that controls its expression.

Shown in Table 3 below are examples of the genes and operons controlledby guanine riboswitches in several bacterial species following abioinformatic analysis involving a sequence homology algorithm asdescribed in Mulhbacher and Lafontaine, 2007. Briefly, theidentification of riboswitches controlling guaA was realized using theRfam database or a published article (Barrick J E and Breaker R R,Genome Biol. 2007) which repertories guanine riboswitches. The NCBIdatabase was then blasted to find the associate genes. Theidentification of new guanine riboswitches in newly sequenced genomescould also be realized using the RNAmotif™ solftware. Genescorresponding to guaA are highlighted. Accession numbers for the genomeof the bacterium and positions corresponding to the beginning of theriboswitch and of the gene or the operon containing guaA are alsopresented. Consistent with the teachings of the present invention,bacterial strains (e.g., S. aureus and C. difficile) expressing the guaAgene under the control of a guanine riboswitch are expected to besusceptible to the antimicrobial effects of the compounds of the presentinvention. Also consistent with the teachings of the present invention,bacterial strains expressing the guaA gene independently from theguanine riboswitch (e.g., E. coli and Bacillus subtilis) are expected tobe not significantly affected by the antimicrobial effects of thecompounds of the present invention.

TABLE 3 Genes and operons controlled by guanine riboswitches in variousbacterial strains guaA or operon guaA or containing guaA operon gene oroperon controlled by guanine containing controlled by guanine guanineriboswitch guaA Bacteria Gram riboswitch riboswitch genome startingending Acholeplasma_laidlawii + uraA Alkaliphilus_metalliredigens + xpt;purE guaA NC_009633 944301 947544 Alkaliphilus_oremlandii + pur operonguaA NC_009922 582562 585305 Bacillus_amyloliquefaciens + xpt, pbuXoperon; pbuG; pur operon Bacillus_anthracis + uraA; GntR; xpt, pbuX guaANC_003997 260657 262446 operon; pur operon; NC_007530 260657 262446 nupCNC_005945 260670 262459 Bacillus_cereus + pbuG; pur operon; xpt, guaANC_012472 272003 273792 pbuX operon; GntR; NC_011658 269262 271050 nupCNC_011773 261264 263053 NC_004722 259617 261405 NC_003909 294525 296313NC_011725 259207 260995 NC_011772 251188 252976 NC_011969 266283 268071NC_006274 265891 267680 NC_009674 271090 272876 Bacillus_clausii + pbuG;pur operon; xpt, pbuX operon Bacillus_halodurans + xpt, pbuX operon; purguaA NC_002570 648458 650224 Operon; uraA Bacillus_licheniformis + puroperon; xpt, pbuX operon; pbuG; nupG Bacillus_pumilus + pbuG; xpt, pbuXoperon; uraA; pur operon Bacillus_subtilis + pbuG; pur operon; yxjA;xpt, pbuX operon Bacillus_thuringiensis + pbuG; pur operon; xpt, guaANC_008600 274659 276448 pbuX operon; GntR; NC_005957 266593 268382 nupCBacillus_weihenstephanensis + uraA; pur operon; guaA NC_010184 262234264023 xpt, pbuX operon; GntR; nupC Bdellovibrio_bacteriovorus −putative secreted nuclease; hypothetical proteinClostridium_acetobutylicum + uraA; xptpbuX operon guaB, guaA NC_0030302824936 2821591 operon Clostridium_beijerinckii + uraA; xptpbuX operonguaB, guaA NC_009617 398946 402351 operon Clostridium_botulinum + uraA;pur operon; pbuX, guaB, guaA NC_009495 3506948 3503782 xpt operon; uraA,apt operon NC_010520 3584820 3581653 operon; uraA NC_009697 34829363479770 NC_009698 3380044 3376878 NC_010516 3567858 3564691 NC_010674396433 399735 NC_010723 391849 395150 NC_009699 3600582 3597415Clostridium_difficile + uraA; xpt; pbuX guaA NC_009089 256232 258074Clostridium_kluyveri + uraA Clostridium_novyi + xpt, pbuG guaB, guaANC_008593 2143257 2139825 operon Clostridium_perfringens + xpt; uraAguaB, guaA NC_003366 2618404 2615047 operon NC_008261 2822240 2818882NC_008262 2482608 2479246 Clostridium_tetani + guaB, guaA NC_0045572551375 2549717 operon Desulfitobacterium_hafniense + purLDesulfotomaculum_reducens + add; uraA, purE operon; uraA;adenylosuccinate lyase Enterococcus_faecalis + xpt, pbuX, pur operonExiguobacterium_sibiricum + xpt, pbuX operon; uraA; guaA NC_010556459675 461411 pur operon Fusobacterium_nucleatum − pur operonGeobacillus_kaustophilus + pbuG; pur operon guaA NC_006510 272489 274168Geobacillus_thermodenitrificans + pbuG; pur operon; xpt, guaA NC_009328252216 253897 pbuX operon Lactobacillus_acidophilus + uraA, pbuG operon;xpt, pbuX operon Lactobacillus_brevis + uraALactobacillus_delbrueckii_bulgaricus + xpt, pbuX operonLactobacillus_fermentum + uraA Lactobacillus_gasseri + uraALactobacillus_helveticus + uraA Lactobacillus_johnsonii + uraALactobacillus_plantarum + uraA; add Lactobacillus_reuteri + uraALactobacillus_salivarius + xpt, pbuX Lactococcus_lactis + xpt, pbuXLeuconostoc_mesenteroides + uraA Listeria_innocua + xpt, pbuX operon;uraA Listeria_monocytogenes + xpt, pbuX operon; uraAListeria_welshimeri + xpt, pbuX operon; uraA Lysinibacillus_sphaericus +uraA, pbuG operon; pur guaA NC_010382 217326 219120 operon; xpt, pbuXoperon; pbuE Oceanobacillus_iheyensis + xpt, pbuX operon guaA NC_004193760489 762329 Oenococcus_oeni + guaB; xpt, pbuX operonPediococcus_pentosaceus + uraA Shewanella_halifaxensis − uraAShewanella_pealeana − uraA Staphylococcus_aureus + xpt, pbuX, guaB,NC_002951 460076 465308 guaA operon NC_009632 466145 471377 NC_009487466075 471307 NC_003923 410563 415795 NC_009782 430774 436006 NC_002758430771 436003 NC_002745 430814 436046 NC_007795 378013 383245 NC_009641425751 430983 NC_007622 383514 388746 NC_007793 436156 441388 NC_010079436051 441283 NC_002952 441067 446299 NC_002953 409208 414440Staphylococcus_carnosus + xpt, pbuX, guaB, NC_012121 46344 51725 guaAoperon Staphylococcus_epidermidis + xpt, pbuX, guaB, NC_004461 24330302427604 guaA operon NC_002976 54833 60259 Staphylococcus_haemoliticus +xpt, pbuX, guaB, NC_007168 2598923 2593514 guaA operonStaphylococcus_saprophyticus + xpt, pbuX, guaB, NC_007350 24076932403978 guaA operon Streptococcus_agalactiae + xpt, pbuX operonStreptococcus_pneumoniae + xpt, pbuX operon Streptococcus_pyogenes +xpt, pbuX operon Streptococcus_thermophilus + xpt, pbuX operonThermoanaerobacter_pseudethanolicus + uraA guaA NC_010321 17605931758904 Thermoanaerobacter_X514 + uraA guaA NC_010320 528317 531544Thermoanaerobacter_tengcongensis + uraA/purE Vibrio_sp − uraAVibrio_splendidus − uraA

FIG. 5 depicts antibiograms showing the effect of compound 1.01 on thegrowth of various bacterial species in which the guanine riboswitchcontrols (FIG. 5B) (i.e. Bh: Bacillus halodurans; STAh: S. haemolyticus;STAa: S. aureus ATCC29213; SA228a: S. aureus resistant to beta-lactams,erythromycin, ciprofloxacin, gentamicin and tetracycline; MRSAcoI:methicilin resistant S. aureus col,; Cb: Clostridium botulinum; Cd6:Clostridium difficile virulent isolated in Quebec; Cd630: Clostridiumdifficile strain 630 with complete sequenced genome; and STAe: S.epidermidis) or not (FIG. 5A) (i.e. Bs: Bacillus subtilis; Ef:Enterococcus feacium; Lm: Listeria monocytogenes; STRd: Streptococcusdysgalactiae; STRpy: Streptococus pyogenes; and STRu: Streptococcusuberis) the expression of the essential guaA gene. Briefly, for eachstrain, bacteria were inoculated at 10⁵ CFU/mL by dilution inMuller-Hinton agar. After agar medium was solidified six wells of 4 mmdiameter were made and filled with 15 μl of tested molecule (5 mg/mL).While strains insensitive to compound 1.01 (shown in FIG. 5A) do nothave the expression of guaA under control of a riboswitch, the strainssensitive to compound 1.01 (shown in FIG. 5B) control guaA expressionvia a riboswitch mechanism.

EXAMPLE 5 Preclusion from Ribosylation of the Compounds of the PresentInvention

Shown schematically in FIG. 6A is the ribosylation reaction leading tothe formation of nucleosides as well as examples of compounds of theinvention that would prevent this ribosylation to occur. Asterisks (*)denote the position within the compound where chemical modificationprecludes ribosylation. FIG. 6B shows typical antibiograms performed asdescribed in the previous example on strains of E. coli ATCC 35695(which lacks a guaA riboswitch) and methicillin resistant S. aureus(which contains a guaA riboswitch) grown in the absence (1) or presenceof 6-thioguanine at a concentration of 0.5 mg/mL (2) or 1 mg/mL (3).FIG. 6C shows that 6-thioguanine is able to be ribosylated and isincorporated into DNA and RNA (Swann et al., 1996), which results in itsriboswitch-independent, non-specific antibiotic activity toward both E.coli and S. aureus (as shown in FIG. 6B).

The effects of several antimicrobial compounds on the growth andsurvival of various strains of E. coli are summarized in Table 4 andpresented in the form of minimum inhibitory concentrations (MIC, inμg/mL). Briefly, minimal inhibitory concentration of compounds 1.01 and1.13 was determined using the microdilution method in 96-wellmicroplates. Bacteria were inoculated at 10⁵ CFU/mL and incubated at 37°C. for 24 h in Muller-Hinton cation adjusted media. Then OD₅₉₅ nm wasread on microplate reader.

TABLE 4 Minimum inhibitory concentrations (MIC) of various antibioticcompounds against E. coli strains E. coli E. coli E. coli CompoundsATCC35695 AcrAB−/− Imp Compound 1.01 >5000 >5000 >5000 Compound1.13 >5000 >5000 >5000 Vancomycin >128 >128 0.25 Erythromicin 1281.0-4.0 0.125-1.0 All concentrations are in μg/mL.

The results depicted in Table 4 above show that the guanine switch-lessbacterium Escherichia coli is not inhibited by compounds 1.01 and 1.13while the E. coli strain is highly permeable (strain E. coli Imp) tolarge antibiotic molecules like erythromycin and vancomycin or lacks theefflux pump AcrAB (strain E. coli AcrAB−/−) that is able to pump toxicmolecules out of the cell. These results demonstrate the antimicrobialspecificity of the compounds of the present invention (e.g., compounds1.01 and 1.13) for bacteria that possess a guanine riboswitchcontrolling the expression of guaA. These results also demonstrate thatthe compounds of the present invention (e.g., compounds 1.01 and 1.13)are not broadly toxic to other bacteria lacking a guanine riboswitchcontrolling the expression of guaA. Thus, the compounds of the presentinvention allow for selective treatment of microbial pathogens affectinganimals and humans. For example, compound 1.01 is sufficiently distinctfrom the natural guanine riboswitch ligand (guanine), to preventincorporation of compounds into cellular nucleosides, nucleotides ornucleic acids, thus preventing toxicity for the mammalian or animal hostin which such a compound is used for therapeutic intervention.

EXAMPLE 6 Minimal Inhibitory Concentrations and Bactericidal Activitiesof Compounds of the Present Invention

As disclosed herein, the compounds of the present invention (e.g.,compounds 1.01 and 1.13) are able to specifically and selectivelyinhibit the growth and/or kill bacterial species that possess theguanine riboswitch controlling the expression of guaA such as S. aureusand C. difficile. The compounds of the present invention demonstrate ananti-bacterial effect on prototypical bacterial strains or bacterialstains causing persistent and chronic bovine mastitis in vitro (e.g., inculture media or milk) and in vivo (e.g., in mice mammary glandinfections). Furthermore, the anti-bacterial effect of the compounds ofthe present invention (e.g., compound 1.01) is as rapid as that of thewell-known antibiotic ciprofloxacin that is used in human and veterinarymedicine.

The effect of various antibiotic compounds, including compounds 1.13 and1.01, on the growth of S. aureus strain ATCC 29213 in vitro inMuller-Hinton cation adjusted media was tested and is shown in FIG. 7and/or Table 5 below. For each experiment, bacteria were inoculated at aconcentration of 10⁵ CFU/mL grown in Muller-Hinton cation adjusted mediain the presence or absence of various antimicrobial compounds. After aspecified time, bacteria were sampling and diluted in 96-wellmicroplates before being inoculated on TSA plate. After 24 hours at 37°C., CFU were counted. FIG. 7A shows the growth of S. aureus strain ATCC29213 as a function of ligand concentration for compounds 1.13 () and1.01 (□). The MICs were determined using the microdilution method in96-well microplates and the bacteria were incubated at 37° C. for 24 hfollowing inoculation. The MIC for compound 1.13 was determined to beabout 5000 μg/mL and that for compound 1.01 was determined to be about600 μg/mL. FIGS. 7B and 7C show the growth of S. aureus strain ATCC29213 as a function of time in the absence () or presence of variouscompounds. Specifically, FIG. 7B shows the anti-bacterial effect of theMIC corresponding to 0.5 μg/mL of erythromycin (▴), 1 μg/mL ofvancomycin (▪), 600 μg/mL of compound 1.01 (▾), and 0.5 μg/mLciprofloxacin (♦). FIG. 7C shows the anti-bacterial effect in theabsence () or presence of the same compounds at a dose of ¼ of theircorresponding MICs (i.e., 0.125 μg/mL ciprofloxacin (♦), 0.125 μg/mLerythromycin (▴), 0.25 μg/mL vancomycin (▪), 150 μg/mL compound 1.01(▾)). FIG. 7D shows the growth of S. aureus strain ATCC 29213 as afunction of time in media alone (), in the presence of 600 μg/mL ofcompound 1.01 (▾), or in the presence of 600 μg/mL of compound 1.01supplemented with 100 μM GMP (i.e. a molecule normally synthesized bythe enzyme GMP synthetase encoded by guaA) (▴).

EXAMPLE 7 Bactericidal Activity of Compound 1.01 on Various S. aureusStrains

The bactericidal activity of compound 1.01 on various strains of S.aureus (including 3 chronic strains and 3 prototypical strains) wasdetermined. These strains include prototypical S. aureus strains ATCC29213, Newbould 305, SHY97-3906 as well as S. aureus strains isolatedfrom persistent and chronic bovine mastitis cases (isolates #3, #557,#1290). C. difficile strain Cd6 was also examined. The bacteria wereinoculated at 10⁵ CFU/mL in Muller-Hinton cation adjusted media inabsence or presence of 600 μg/mL compound 1.01 and grown for 4 hours.The results are summarized in Table 5 below and are expressed as themean fold reduction of CFU/mL (i.e., the number of CFU/mL of controlbacteria divided by the number of CFU/mL of treated bacteria). Theseresults indicate that compound 1.01 has a significant anti-bacterialeffect against all the strains tested, with the highest effect beingagainst S. aureus strain ATCC 29213.

TABLE 5 Bactericidal activity of compound 1.01 on S. aureus and C.difficile strains Fold reduction of CFU/mL (control/treated) StrainsMean (log10) SD S. aureus ATCC 29213 6.67 0.58 S. aureus Newbould 3054.86 1.42 S. aureus Chronic #3 5.11 1.25 S. aureus Chronic #1290 5.421.56 S. aureus Chronic #557 4.38 1.86 S. aureus SHY97-3906 6.35 1.26 C.difficile (Cd6) 5.42 1.02

EXAMPLE 8 Mode of Action of Compounds of the Present Invention

To verify the specificity and explore the mechanism of action ofcompound 1.01, a transcriptomic microarray analysis was used to followthe expression of more then 500 S. aureus genes. Compound 1.01 was usedalone or in combination with GMP and the results are shown in FIG. 8A.Briefly, the relative expression of S. aureus genes (xpt, pbuX, guaA andguaB) under the control of guanine riboswitch when grown in presence ofcompound 1.01 or compound 1.01 supplemented with GMP was examined.Bacteria were inoculated at 10⁸ CFU/mL in Muller-Hinton cation adjustedmedia in absence or presence of 600 μg/mL of compound 1.01 or 600 μg/mLcompound 1.01 supplemented with 100 μM GMP. After 30 min of growth, RNAwas extracted and 2.5 μg of RNA were submitted to reverse transcriptionto generate fluorescent probes through an aminoallyl cDNA labelingprocedure before being hybridized on the microarray (Moisan et al.,2006). The experiments were repeated three times and the averages andstandard deviations are shown. Gene expression was normalized to that ofxpt in the presence of compound 1.01. The expression levels the genesgyrA and gyrB (encoding gyrase protein subunits) were also included ascontrols since their expression is not down-regulated in the presence ofGMP. When compound 1.01 was used alone, more then 75% of genes wererepressed including those controlled by the guanine riboswitch,xpt/pbuX/guaB/guaA (FIG. 8A). This could be explained by the inhibitionof GMP production that is essential for the synthesis of messenger RNA(see FIG. 8B). When compound 1.01 and GMP were added in combination, theGMP allowed most of the studied genes to maintain their expression leveland only 20% genes were repressed, among which were those controlled bythe guanine riboswitch, xpt/pbuX/guaB/guaA (FIG. 8A). As depicted inFIG. 8B, the compounds of the present invention (e.g., compound 1.01)can interact with the guanine riboswitch and reduce expression of thegenes xpt/pbuX/guaB/guaA. In particular, the inhibition of guaA causes acellular depletion of the levels of GMP, which in turn inhibits generalDNA and RNA synthesis. The supplementation of GMP counteract the effectsof the compounds of the present invention (e.g., compound 1.01) on DNAand RNA synthesis. These results demonstrate the specificity of compound1.01 as shown on the schema of FIG. 8B.

FIG. 8B shows a schematic representation of the inhibitory effect ofcompound 1.01 in S. aureus. A unique guanine riboswitch performs geneexpression regulation of all four guanine-related genes (xpt, pbux, guaAand guaB). Riboswitch-regulated genes appear in gray and the compound1.01 inhibitory effect is indicated by grey bars. A thick bar indicatesthe effect of compound 1.01 on expression of guaA. Broken arrowsrepresent multiple synthesis steps that are not shown in the figure.Ovals represent genes encoding various metabolite transporters.

EXAMPLE 9 Inhibitory Effect of Compounds of the Present Invention DuringIn Vivo Infection in Mice and Cows

The compounds of the present invention (e.g., compound 1.01) are able toinhibit the growth and/or kill microbial pathogens possessing theguanine riboswitch controlling the expression of guaA during infectionof the mammary glands in the mouse.

The efficacy of compound 1.01 in treating a S. aureus infection of themouse mammary glands was examined. Briefly, mouse mammary glands wereinfected with 100 CFU of S. aureus. Four hours after the inoculation,the glands were treated with vehicle (PBS), 10, 50 or 100 μg/gland ofcompound 1.01. Six hours later, glands were excised, homogenized andplated on Mueller-Hinton agar. CFU were then counted after 18 hours at35° C. The results of these studies are shown in FIG. 9. Each dotrepresents the CFU of each individual gland (n=6-12) and the medianvalue for each group is indicated by the bar. Statistical differences (P<0.05) between CFU recovered from treated and untreated animals areshown by asterisks (non-parametric Kruskal-Wallis ANOVA with Dunn's posttest). These results clearly demonstrate the anti-microbial effect invivo of compound 1.01 against a S. aureus infection. This demonstratesthat a compound such as compound 1.01 is sufficiently soluble, stableand bioavailable to display anti-microbial activity during an infectionin an animal host. Intra-mammary injection of 10 times the effectivedose of a compound such as compound 1.01 is able to reduce bacterialcounts by >3 log₁₀ without causing any significant signs of toxicity inmice, such as alterations in posture, breathing, piloerection ormovement. There was also no apparent cytotoxicity upon histologicalobservations of mammary tissues in 1.01-treated mice (100 μg/gland)compared to PBS-treated glands. Similarly, in tests performed in 4Holstein cows over a period of 48 h, the intramammary injection ofcompound 1.01 at either 0 (saline control), 100 mg, 250 mg or 500 mg inthe 4 individual quarters caused no change in body temperature, milkappearance, milk somatic cell counts or individual milk quarterproduction that could be correlated with any of the treatment.

EXAMPLE 10 Compounds of the Present Invention Prevent Rapid Developmentof Bacterial Resistance in S. aureus

The present invention relates to the discovery that the compounds of thepresent invention are not only able to inhibit the expression of thegene guaA when it is under riboswitch control, but can also preventdevelopment of bacterial resistance. Specifically, FIG. 10 shows theinability of S. aureus to develop resistance toward compound 1.01.Briefly, serial passages in the presence of sub-inhibitoryconcentrations of test antibiotics demonstrating the inability of S.aureus to develop resistance toward 1.01 were conducted. The MIC of testcompounds against S. aureus strain ATCC 29213 recovered from brothcultures containing sub-inhibitory concentrations of antibiotics wasdetermined every 5 passages up to 30. As a comparison, results obtainedwith two known antibiotics, ciprofloxacin and rifampicin, were added tothe histogram. High level resistance to ciprofloxacin and rifampicin wasrapidly selected (within 5 daily passages) in S. aureus. Such rapiddevelopment of resistance for the traditional drugs is consistent withthe selection of known single point mutations each able to provide adecrease in drug affinity for the bacterial cell target. There are atleast 2 known point mutations in GyrA conferring resistance tociprofloxacin in addition to possible over-expression of the NorA effluxpump system also occurring through mutations (at least 3 possiblemutations) (Jones et al., 2000) and at least 17 possible differentmutations in RpoB enabling resistance to rifampicin have been documented(Wilchelhaus et al., 2001). The S. aureus strain did not developantibiotic resistance to compound 1.01 after at least 30 passages inculture. The absence of resistance observed in presence of 1.01 isprobably because reestablishing guaA gene expression in the presence of1.01 requires multiple mutational steps thus reducing the frequency ofresistance development and/or that maintaining a functional riboswitchis a vital process that does not allow bacteria to bypass 1.01antibiotic action. These results also strongly suggest that thecompounds of the present invention not only possess an anti-microbialeffect, but may also prevent the development of multi-drug resistantbacteria.

EXAMPLE 11 Illustrative Compounds of the Present Invention

The present invention relates to a number of small molecule compoundshaving a selective antimicrobial activity on microbial pathogenscontaining guaA under control of the guanine riboswitch but having nosubstantial antimicrobial activity against microorganisms lacking aguaA-controlled by the guanine riboswitch. The compounds of the presentinvention fit the structural requirements for binding to the guanineriboswitch binding site and yet are chemically unable to be ribosylatedby the targeted pathogens. The latter is meant at preventingincorporation of the compounds into cellular nucleosides, nucleotides ornucleic acids, and thus preventing broad, non-specific toxicity for themammalian or animal host in which the present compounds are used fortherapeutic intervention.

Representative structures of the compounds of the present inventionderived from formulas 1.0, 2.0 and 3.0 are shown in FIG. 11. All thesecompounds are lacking the required chemistry to undergo ribosylation bybacterial or mammalian enzymes which could lead to broad andnon-specific toxicity.

EXAMPLE 12 Effect of Compounds of the Present Invention on the Treatmentof Infections

In addition to intramammary infections, S. aureus is a pathogen alsofound in skin and skin structure infections, respiratory tractinfections, blood and urine (Garau et al., 2009; Araki et al, 2002;Corey, 2009). Clostridium infections in humans such as those caused byC. difficile, C. botulinum, C. tetani and C. perfringens (Leffler andLamont, 2009; Khanna, 2008), in poultry by C. perfringens (Van Immerseelet al., 2004) and in pigs are also of importance and the colonization ofpigs by MRSA is also a problem linked to human health (Songer and Uzal,2005; Khanna et al., 2008). The invention can therefore also be used fortreatment of a variety of clostridial or staphylococcal infectionsoccurring at body sites other than the mammary glands in animals and inhumans.

EXAMPLE 13 Aspects Relating to Synthesis of Compounds of the PresentInvention

Analogue A can be synthesized from chloronitropyrimidine (A1) (Patel etal., 2007). Synthesis of A1 as described by Sircar et al., 1986 to givethe precursor aminomercaptopyrimidine (A2). The latter can then bederivatized with ethyl oxoacetate to give the desired product A (R═H).This scheme will allow additional functionalization as well.

Pyridopyrimidine B can be synthesized following a procedure reported byUrleb et al., 1990 for a similar analogue. Pterin (X═N) is commerciallyavailable.

Oxazole derivative D was synthesized similarly to the synthesis of amethylated analogue (Miller et al, 2002).

Pyrrole derivative E can be synthesized as described by Taylor andYoung, 1995.

EXAMPLE 14 Bio-Informatics Search of Guanine Riboswitches.

Natural riboswitches contain aptamer domains which are typically veryconserved in their sequence and structure given that they must bindcellular metabolites that are conserved through evolution. These aptamerdomains exhibit a conserved scaffold of base-paired helices thatorganize the overall fold of each aptamer. The identities of each basein these helices most often reflect their need to be base-paired but incontrast, the identities of bases that are in direct contact with thebound metabolite are very highly conserved given precise role in theformation of the ligand binding site. Thus, it is possible to define aconsensus sequence, representing all possible aptamer sequences, whichcan be used to perform computational searches in biological sequencedatabases. For instance, by translating the consensus sequence into analgorithm search, the software RNAmotif™ (Macke et al., 2001) can screenfor all aptamer occurrences in the RefSeq™ microbial database (Pruitt etal., 2005). The output of the software gives genomic locations of allaptamer found. There is already a subgroup of aptamer sequences forvarious riboswitch families that have been retrieved by the scientificcommunity that can be found at the Rfam™ database (Griffiths-Jones etal., 2005), but because it was observed that it is only partiallycomplete, it was found preferable to build a database using RNAmotif™.

Below is an example of a consensus sequence used for the bioinformaticsearch. The meaningfulness of the characters and commands described inthe consensus are very well described in the original RNAmotif™ article(Macke et al., 2001). This corresponds to possible guanine riboswitchesthat could adopt a similar structures than the known guanine riboswitch.

parms wc += gu; descr h5( minlen=4, maxlen=10, mispair=0, ends=‘mm’) ss( seq=“{circumflex over ( )}UA.\{0,1\}$”) h5( minlen=5, maxlen=8,mispair=3, ends=‘mm’) ss ( seq=“{circumflex over( )}.\{0,1\}AU.\{0,3\}GG$” ) h3 ss ( seq=“{circumflex over( )}GNNNCUAC$” ) h5( minlen=5, maxlen=8, mispair=2, ends=‘mm’ ) ss (seq=“{circumflex over ( )}CC.\{0,3\}A.\{0,1\}$”) h3 ss (seq=“{circumflex over ( )}N\{0,1\}C.\{0,1\}$”) h3 H, Hoogsteen face; MI,mutual information; nt, nucleotides;

EXAMPLE 15 Ability of Compounds of the Invention to Inhibit S. aureusGrowth

As disclosed herein, the compounds of the present invention (e.g.,compounds 1.16, 2.02 and 2.17) (1.16 and 2.17 were purchased fromSigma-Aldrich and Toronto Research Chemical respectively) are able tospecifically and selectively inhibit the growth and/or kill bacterialspecies that possess the guanine riboswitch controlling the expressionof guaA such as S. aureus. As reported in Table 6 (below), compounds1.16, 2.02 and 2.17 are able to bind guanine riboswitch and to inhibitS. aureus growth with a MIC of 0.128, 1.024 and 0.128 mg/mL,respectively.

The effect of the antibiotic compounds of formula 2.0, includingcompounds 2.02 and 2.17, on the growth of S. aureus strain ATCC 29213 invitro in Muller-Hinton cation adjusted media allow to confirm theefficiency of compounds of general formula 2.0 to achieve antibioticactivity. For each compound, antibiotic activity was also tested on E.coli strain ATCC 35695 and no antimicrobial activity of the compounds(1.16, 2.02 and 2.17) was observed. The MICs were determined using themicrodilution method in 96-well microplates and the bacteria wereincubated at 37° C. for 24 h following inoculation.

As required, the compounds of Table 6 present no ribosylation site, sothis will prevent their incorporation in DNA and their mutagenicpotential.

TABLE 6 Description of the properties of various guanine riboswitchligands Guanine S. aureus riboswitch growth Appropriate CompoundStructure binding inhibition Ribosylation properties 1.16

Yes Yes MIC 0.128 mg/mL No Yes 2.02

Yes Yes MIC    1 mg/mL No Yes 2.17

Yes Yes MIC 0.128 mg/mL No Yes

EXAMPLE 16 Route to afford compound 2.02:5-Amino-6H-oxazolo[5,4-d]pyrimidin-7-one

5-Formamino-2-amino-4,6-dihydroxypyrimidine

Sodium pieces (242 mg, 10.5 mmol) were added to MeOH (20 mL). After allthe sodium had reacted, guanidine hydrochloride (1.00 g, 10.5 mmol) wasadded and the reaction mixture was stirred for 15 min. The reactionmixture was then heated to reflux and during this time, diethylacetamidomalonate (2.13 g, 10.5 mmol) was added. After 2 h, more MeOH (20 mL) wasadded and the reaction mixture was refluxed overnight. The reactionmixture was then cooled to rt, filtered, washed with MeOH and chloroformand dried under vacuum at 50° C. The resulting solid was dissolved inwater (25 mL) and was precipitated by the addition of 50% hydrochloricacid. The solution was filtered and the solid was washed with water andacetone and was finally dried under vacuum at 50° C. to afford compound7 as a white solid (900 mg, 50%). mp>300° C. ¹H NMR (300 MHz, D₂O) δ(ppm) 8.19 (s, 1H). ¹³C NMR (75.5 MHz, DMSO-d₆) δ (ppm) 174.9, 168.2,154.5, 92.6, 21.9.

5-Amino-6H-oxazolo[5,4-d]pyrimidin-7-one

Compound 7 (125 mg, 0.73 mmol) was dissolved in 12 M H₂SO₄ (1.50 mL)using an ultrasonic bath. The dark yellow solution was stirred at rtovernight. THF (5 mL) was then added at 0° C. to form a yellowprecipitate. The mixture was stored at 4° C. for 2 h and was centrifugedto obtain compound 8 as a yellow solid. ¹H NMR (300 MHz, DMSO-d₆) δ(ppm) 8.71 (br s, 1H), 7.45 (br s, 1H). ¹³C NMR (75.5 MHz, DMSO-d₆) δ(ppm) 158.6, 152.3, 84.2. LRMS (m/z, relative intensity) 137 (MH⁺—NH₃,100), 153 (MH⁺, 50), 159 (MNa⁺—NH₃, 90). HRMS calculated for C₆H₆N₄O₃:153.0413, found: 152.9617.

Although the present invention has been described hereinabove by way ofspecific embodiments thereof, it can be modified, without departing fromthe spirit and nature of the subject invention as defined in theappended claims.

REFERENCES

-   Anderson, V. E., R. P. Zaniewski, F. S. Kaczmarek, T. D. Gootz,    and N. Osheroff. 2000. Action of quinolones against Staphylococcus    aureus topoisomerase IV: Basis for DNA cleavage enhancement.    Biochem. 39:2726.-   Anderson et al, The Practice of Medicinal Chemistry (1996), Academic    Press, New York; and in The Orange Book (Food & Drug Administration,    Washington, D.C. on their website).-   Araki, M., R. Kariyama, K. Monden, M. Tsugawa, and H. Kumon. 2002.    Molecular epidemiological studies of Staphylococcus aureus in    urinary tract infection. J. Infect. Chemother. 8:168-174.-   Barrick, J. E. and R. R. Breaker (2007) The distributions,    mechanisms, and structures of metabolite-binding riboswitches,    Genome Biol 8, R239.-   Batey, R. T., S. D. Gilbert and R. K. Montange (2004) Structure of a    natural guanine-responsive riboswitch complexed with the metabolite    hypoxanthine, Nature 432, 411.-   Berge, S. M., Bighley, L. D., Monkhouse, D. C. (1977) Pharmaceutical    salts. J Pharm Sci. 66:1-19.-   Blount K. F. and Breaker R. R. (2006) Riboswitches as antibacterial    drug targets. Nature Biotechnology 24 :1558-1564.-   Coppins, R. L., Hall, K. B., Groisman, E. A. (2007) The intricate    world of riboswitches. Curr Opin Microbiol. 10(2):176-181.-   Corey, G. R. 2009. Staphylococcus aureus bloodstream infections:    Definitions and treatment. Clin. Infect. Dis. 48 (suppl.    4):5254-5259.-   Garau, J., E. Bouza, J. Chastre, F. Gudiol, and S. Harbarth. 2009.    Management of methicillin-resistant Staphylococcus aureus    infections. Clin. Microbiol. Infect. 15:125-136.-   Griffiths-Jones, S., Moxon, S., Marshall, M., Khanna, A., Eddy, S.    R., and Bateman, A. (2005) Rfam: annotating non-coding RNAs in    complete genomes, Nucleic Acids Res. 33, D121-4.-   Macke T J, Ecker D J, Gutell R R, Gautheret D, Case D A, Sampath R:    RNAMotif, an RNA secondary structure definition and search    algorithm. Nucleic Acids Res 2001, 29:4724-4735.-   Jones, M. E., N. M. Boenink, J. Verhoef, K. Kohrer and F.-J.    Schmitz. 2000. Multiple mutations conferring ciprofloxacin    resistance in Staphylococcus aureus demonstrate long-term stability    in an antibiotic-free environment. J. Antimicrob. Chemother.    45:353-356.-   Khanna, N. 2008. Clindamycin-resistant Clostridium perfringens    cellulitis. J. Tissue Viability 17:95-97.-   Khanna, T., R. Friendship, C. Dewey, J. S. Weese. 2008. Methicillin    resistant Staphylococcus aureus colonization in pigs and pig    farmers. Vet. Microbiol. 128:298-203.

Knowles D. J., Foloppe N., Matassova N. B., Murchie A. I. The bacterialribosome, a promising focus for structure-based drug design. Currentopinion in pharmacology 2002. 2:501-502.

-   Leffler, D. A., and J. T. Lamont. 2009. Treatment of Clostridium    difficile-associated disease. Gastroenterology 136:1899-1912.-   Mayer, S. J., A. E. Watennan, P. M. Keen, N. Craven, and F. J.    Bourne (1988) Oxygen concentration in milk of healthy and mastitic    cows and implications of low oxygen tension for the killing of    Staphylococcus aureus by bovine neutrophils. J. Dairy Res. 55:513.-   Miller, D. J., Ravikumar, K., Shen, H., Suh, J.-K., Kerwin, S. M.,    and Robertus, J. D. (2002) J. Med. Chem. 45:90-98.-   Moisan, H., E. Brouillette, C. L. Jacob, P. L. Begin, S. Michaud,    and F. Malouin. 2006. The Transcription of Virulence Factors in    Staphylococcus aureus Small Colony Variants Isolated from Cystic    Fibrosis Patients is Influenced by SigB. J. Bacteriol. 188:64-76.-   Mulhbacher, J. and D. A. Lafontaine (2007) Ligand recognition    determinants of guanine riboswitches. Nucleic Acids Res 35 :5568.-   Patel, P. R., Ramalingan, C., Park, Y. T. (2007) Synthesis and    antimicrobial evaluation of guanylsulfonamides. Bioorg Med Chem    Lett. (2007) 17:6610-4.-   Pruitt, K., Tatusova, T., and Maglott, D. 2005. NCBI Reference    Sequence (RefSeq): A curated nonredundant sequence database of    genomes, transcripts, and proteins. Nucleic Acids Res. 33:    D501-D504. doi: 10.1093/nar/gki011.-   Russo, T. A., S. T. Jodush, J. J. Brown, and J. R. Johnson. (1996)    Identification of two previously unrecognized genes (guaA and argC)    important for uropathogenesis. Mol. Microbiol. 22:217-229.-   Samant, S., H. Lee, M. Ghassemi, J. Chen, J. L. Cook, A. S. Mankin,    and A. A. Neyfakh. (2008) Nucleotic biosynthesis is crucial for    growth of bacteria in human blood. PloS Pathogens 4(2):e37    (0001-0010).-   Sears, P. M. and K. K. McCarthy (2003) Management and treatment of    staphylococcal mastitis. Vet. Clin. Food Anim. Pract. 19:171-185.-   Sircar, J. C., Suto, M. J., Scott, M. E., Dong, M. K.,    Gilbertsen, R. B. (1986) Inhibitors of human purine nucleoside    phosphorylase. Synthesis, purine nucleoside phosphorylase    inhibition, and T-cell cytotoxicity of    2,5-diaminothiazolo[5,4-d]pyrimidin-7(6H)-one and    2,5-diaminothiazolo[4,5-d]pyrimidin-7(6H)-one. Two thio isosteres of    8-aminoguanine. J Med Chem. 1986 September; 29(9):1804-6.-   Songer, J. G., and F. A. Uzal. 2005. Clostridial enteric infections    in pigs. J. Vet. Diagn. Invest. 17:528-536.-   Swann, P. F., Waters, T. R., Moulton, D. C., Xu, Y. Z., Zheng, Q.,    Edwards, M., Mace, R. Role of postreplicative DNA mismatch repair in    the cytotoxic action of thioguanine. (1996) Science.    273(5278):1109-11.-   Talbot G H, Bradley J, Edwards J E Jr, Gilbert D, Scheid M, Bartlett    J G; Antimicrobial Availability Task Force of the Infectious    Diseases Society of America. Clin Infect Dis. 2006 42:657-658-   Taylor, E. C., Young, W. B. (1995) Pyrrolo[3,2-d]pyrimidine folate    analogues: “inverted” analogues of the cytotoxic agent LY231514. J    Org Chem. 60:7947-52.-   Tiedeman, A., J M Smith and H Zalkin. 1985. Nucleotide Sequence of    the guaA gene encoding GMP synthetase of Escherichia coli K12. J.    Biol. Chem. 260:8676-79.-   Urleb, U., Stanovnik, B., Tisler, M. (1990) The Synthesis and    Transformations of 2-Ethoxycarbonyl-3-Isothiocyanatopyridine.    Pyrido[3,2-d]pyrimidines and some Azolopyrido[3,2-d]pyrimidines. J.    Het. Chem. 27:407-12.-   Van Immerseel, F., J. De Buck, F. Pasmans, G. Huyghebaert, F.    Haesebrouck, and R. Ducatelle. 2004. Clostridium perfringens in    poultry: an emerging threat for animal and public health. Avian    Pathol. 33:537-549.-   Wilchelhaus, T. A., V. Schafer, V. Brade and B. Boddinhaus. 2001.    Differential effect of rpoB mutations on antibacterial activities of    rifampicin and KRM-1648 against Staphylococcus aureus. J.    Antimicrob. Chemother. 47:153-156.-   WO 2006/055351, Breaker R. et al., 2006.

1. A compound of the general formula 1.0,

wherein, when said compound is bound to a guanine riboswitch, R3 mayserve as a hydrogen bond acceptor but cannot be ribosylated; wherein

represents a single or double bond; wherein R3 is —N═, —S—, —CH— or —O—;wherein R6 is a carbon atom; wherein R1, R2, R4 or R5 are identical ordifferent and are independently —NR8-, —CHR8-, ═CR8-, —O(═O)— or—C(═NR8)-; wherein R8 is —H, —NH₂, —OH, —SH, a halide, fluorine,chlorine, bromine, iodine, —CO₂H, —CO₂-alkyl, —CO₂-aryl, —C(O)NH₂, —CH₃,—POOH, —SO₂alkyl, —SO₂aryl, —SH, substituted or unsubstituted alkyl,substituted or unsubstituted alkoxy, substituted or unsubstituted aryl,substituted or unsubstituted aryloxy, substituted or unsubstitutedbenzyloxy, substituted or unsubstituted —NHalkyl, substituted orunsubstituted —NHalkoxy, -substituted or unsubstituted NHC(O)alkyl,substituted or unsubstituted —NHCO₂alkyl, substituted or unsubstituted—NH—NHalkyl, substituted or unsubstituted NH—NHalkoxy, substituted orunsubstituted —NH—SO₂alkyl, —NHC(O)NH₂, —NH—NH₂, —NH—SO₂—R9,—NHCO₂CH₂—R9, —NH—OR9, —NH₂—R9, —NH—NH—R9, —NHR9, —NH—CH₂—R9, or—NH—NH—CH₂—R9, wherein R9 is:

wherein n is an integer from 1 to 5; wherein R10 is —H, —NH₂, —OH,alkoxy, —N-morpholino, a halide, fluorine, chlorine, bromine or iodine;wherein, R7 is ═O, —OH, —SH, —NH₂, —CO₂H, —CO₂-alkyl, —CO₂-aryl,—C(O)NH₂, -alkoxy, -aryloxy, -benzyloxy, a halide, fluorine, chlorine,bromine, iodine, —NHalkyl, —NHalkoxy, —NHC(O)alkyl, —NHCO₂alkyl, ═NR8,═NR9, —NHCO₂CH₂—R9, —NHC(O)NH₂, —NH—NH₂, —NH—NHalkyl, —NH—NHalkoxy,—SO₂alkyl, —SO₂aryl, —NH—SO₂alkyl, —NH—SO₂—R9, —NH—OR9, —NH—R9,—NH—NH—R9, —NH—NH—CH₂—R9, or —NH—CH₂—R9, wherein R8 and R9 are asdefined above, with the proviso that the compound is not:4-hydroxy-2,5,6-triaminopyrimidine (1.01);2,4-diamino-6-hydroxypyrimidine (1.13); or4,5-diamino-6-hydroxy-2-mercaptopyrimidine (1.16).
 2. A compound of thegeneral formula 2.0,

wherein, when said compound is bound to a guanine riboswitch, R9 mayserve as a hydrogen bond donor but cannot be ribosylated; wherein

represents a single or double bond; wherein R3 is —N═, —S—, or —O—;wherein R1 or R2 are identical or different and are independently—NR11-, —CHR11-, ═CR11-, —C(═O)— or —C(═NR11)-; wherein R11 is —H, —NH₂,—OH, —SH, —CO₂H, —CO₂-alkyl, —CO₂-aryl, —C(O)NH₂, —CH₃, —POOH,—SO₂alkyl, —SO₂aryl, —SH, substituted or unsubstituted alkyl,substituted or unsubstituted alkoxy, substituted or unsubstituted aryl,substituted or unsubstituted aryloxy, substituted or unsubstitutedbenzyloxy, substituted or unsubstituted —NHalkyl, substituted orunsubstituted —NHalkoxy, substituted or unsubstituted —NHC(O)alkyl,substituted or unsubstituted —NHCO₂alkyl, substituted or unsubstituted—NH—NHalkyl, substituted or unsubstituted —NH—NHalkoxy, substituted orunsubstituted —NH—SO₂alkyl, —NHC(O)NH₂, —NH—N H₂, —NH—SO₂—R12,—NHCO₂CH₂—R12, —NH—OR12, —NH₂—R12, —NH—NH—R12, —NHR12, —NH—CH₂—R12, or—NH—NH—CH₂—R12; wherein R12 is:

wherein n is in integer from 1 to 5; wherein R13 is at least one of —H,—NH₂, —OH, alkoxy, —N-morpholino, a halide, fluorine, chlorine, bromine,iodine; wherein R4, R5 and R6 are carbon atoms; wherein R7 is —═N—,—NH—, —CH₂—; —O— or —S—; wherein R8 is —CH₂, —O—, —S—, —CHR13- or—CR13=, wherein R13 is as defined above; wherein R9 is —CH₂—; —O—, —S—,or —P(OOH)—, —N═; wherein R10 is ═O, —OH, —SH, —NH₂, —CO₂H, —CO₂-alkyl,—CO₂-aryl, —C(O)NH₂, -alkoxy, -aryloxy, -benzyloxy, a halide, fluorine,chlorine, bromine, iodine, —NHalkyl, —NHalkoxy, —NHC(O)alkyl,—NHCO₂alkyl, ═NR11, ═NR12, —NHCO₂CH₂—R12, —NHC(O)NH₂, —NH—NH₂,—NH—NHalkyl, —NH—NHalkoxy, —SO₂alkyl, —SO₂aryl, —NH—SO₂alkyl,—NH—SO₂—R12, —NH—OR12, —NH—R12, —NH—NH—R12, —NH—NH—CH₂—R12, or—NH—CH₂—R12; wherein R11 and R12 are as defined above, with the provisothat the compound is not: guanine, hypoxanthine, xanthine or5-amino-2-chloro-2,3-dihydrothiazolo[4,5]pyrimidine-7-(6H)-one (2.17).3. A compound of the general formula 3.0,

wherein, when the compound is bound to a guanine riboswitch, R10 mayserve as a hydrogen bond donor but cannot be ribosylated; wherein

represents a single or double bond; wherein R3 is —N═, —S—, or —O—;wherein R10 is —N═, —CH₂—; —O—, —S—, or —P(OOH)—; wherein R1, R2 andR9are identical or different and are independently —NR12-, —CHR12-,═CR12-, —O(═O)— or —C(═NR12)-; wherein R12 is —H, —NH₂, —OH, —SH, —CO₂H,—CO₂-alkyl, —CO₂-aryl, —C(O)NH₂, —CH₃, —POOH, —SO₂alkyl, —SO₂aryl, —SH,substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy,substituted or unsubstituted aryl, substituted or unsubstituted aryloxy,substituted or unsubstituted benzyloxy, -substituted or unsubstitutedNHalkyl, substituted or unsubstituted —NHalkoxy, substituted orunsubstituted —NHC(O)alkyl, substituted or unsubstituted —NHCO₂alkyl,substituted or unsubstituted —NH—NHalkyl, substituted or unsubstituted—NH—NHalkoxy, substituted or unsubstituted —NH—SO₂alkyl, —NHC(O)NH₂,—NH—NH₂, —NH—SO₂—R13,—NHCO₂CH₂—R13, —NH—OR13, —N+H2-R13, —NH—NH—R13,—NHR13, —NH—CH₂—R13, or —NH—NH—CH₂—R13; wherein R13 is:

wherein n is an integer from 1 to 5; wherein R14 is —H, —NH₂, —OH,alkoxy, —N-morpholino, a halide, fluorine, chlorine, bromine, or iodine;wherein R4, R5 and R6 are carbon atoms; wherein R7 and R8 are identicalor different and are independently —CH═N—, —CH₂—O—, CH₂—S—, or CH₂SO₂—;wherein R11 is ═O, —OH, —SH, —NH₂, —CO₂H, —CO₂-alkyl, —CO₂-aryl,—C(O)NH₂, -alkoxy, -aryloxy, -benzyloxy, a halide, fluorine, chlorine,bromine, iodine, —NHalkyl, —NHalkoxy, —NHC(O)alkyl, —NHCO₂alkyl, ═NR12,═NR13, —NHCO₂CH₂—R13, —NHC(O)NH₂, —NH—NH₂, —NH—NHalkyl, —NH—NHalkoxy,—SO₂alkyl, —SO₂aryl, —NH—SO₂alkyl, —NH—SO₂—R13, —NH—OR13, —NH—R13,—NH—NH—R13, —NH—NH—CH₂—R13, or —NH—CH₂—R13; and wherein R12 and R13 areas defined above.
 4. The compound of claim 2, wherein: (i) R1 is —NH—;(ii) R2 is ═CNH₂—; (iii) R3 is —N═; (iv) R7 is —N═ or —S—; (v) R8 is—CR13=; (vi) R9 is —O— or —N═; (vii) R10 is ═O or ═NH; or (viii) anycombination of (i) to (vii).
 5. The compound of claim 2, wherein saidcompound is of the general formula 2.0a,

wherein R2, R7, R8, R9 and R10 are as defined in claim
 2. 6. Thecompound of claim 5, wherein R2 is ═CNH₂—.
 7. The compound of claim 5,wherein R7 is —N═ or —S—.
 8. The compound of claim 5, wherein R8 is—CR13=.
 9. The compound of claim 5, wherein R9 is —O— or —N═.
 10. Thecompound of claim 5, wherein R10 is ═O or ═NH.
 11. A compound of thegeneral formula 3.0a,

wherein R2, R7, R10 and R11 are as defined in claim
 3. 12. The compoundof claim 1, wherein (i) R1 is —NH—; (ii) R2 is —C(NH₂)═ or ═C(SH)—;(iii) R3 is —N═; (iv) R4 is ═CNH₂—; (v) R5 is —CH═ or —C(NH₂)═; (vi) R6is —C═; (vii) R7 is ═NH or ═O; or (viii) any combination of (i) to(vii).
 13. The compound of claim 1, wherein said compound is of thegeneral formula 1.0a,

wherein R2 and R7 are as defined in claim 1, wherein R5 is —CH═ or—C(NH₂)═, and wherein R4 is —C(NH₂)═.
 14. The compound of claim 13,wherein R7 is ═O or ═NH.
 15. The compound of claim 13, wherein R2 is—C(NH₂)═ or —C(SH)═.
 16. A composition comprising the compound definedin claim 1, and (a) an antibiotic; (b) an antiseptic; (c) adisinfectant; (d) a diluent; (e) an excipient; (f) a pharmaceuticallyacceptable carrier; or (g) any combination of (a)-(f).
 17. A compositioncomprising (i) 4-hydroxy-2,5,6-triaminopyrimidine (formula 1.01); (ii)4,5-diamino-6-hydroxy-2-mercaptopyrimidine (formula 1.16) (iii)2,4-diamino-6-hydroxypyrimidine (formula 1.13); or (iv)5-amino-2-chloro-2,3-dihydrothiazolo[4,5]pyrimidine-7-(6H)-one (2.17);and (a) an antibiotic; (b) an antiseptic; (c) a disinfectant; (d) apharmaceutically acceptable carrier; or (e) any combination of (a)-(d).18. The composition of claim 16, wherein said composition is apharmaceutical composition.
 19. A method of preventing or treating amicrobial infection in a subject, said method comprising administeringto said subject a therapeutically effective amount of a non ribosylableligand of a guanine riboswitch, wherein said microbial infection iscaused by a pathogen bearing the guanine riboswitch, and wherein theguanine riboswitch controls the expression of guaA.
 20. The method ofclaim 19, wherein said non ribosylable ligand of a guanine riboswitch is(i) the compound defined in claim 1; (ii)4-hydroxy-2,5,6-triaminopyrimidine (formula 1.01); (iii)4,5-diamino-6-hydroxy-2-mercaptopyrimidine (formula 1.16); (iv)2,4-diamino-6-hydroxypyrimidine (formula 1.13); or (v)5-amino-2-chloro-2,3-dihydrothiazolo[4,5]pyrimidine-7-(6H)-one (2.17).21. The method of claim 19, wherein said subject is an animal.
 22. Themethod of claim 21, wherein said subject is a cow or a human.
 23. Themethod of claim 19, wherein said pathogen is a bacteria belonging to thegenus Staphylococcus or Clostridium.
 24. The method of claim 23, whereinsaid bacteria is Staphylococcus aureus, methicillin-resistantStaphylococcus aureus, Staphylococcus epidermidis, Staphylococcushaemolyticus, Clostridium botulinum or Clostridium difficile.
 25. Themethod of claim 19, wherein said infection is a mammary gland infection.26.-33. (canceled)
 34. A method of disinfecting and/or sterilizing anobject of a pathogen, said method comprising applying an effectiveamount of a non ribosylable ligand of a guanine riboswitch to saidobject, wherein said pathogen bears a guanine riboswitch that controlsthe expression of guaA.
 35. The method of claim 34, wherein said nonribosylable ligand of a guanine riboswitch is (i) the compound definedin claim 1; (ii) 4-hydroxy-2,5,6-triaminopyrimidine (formula 1.01);(iii) 4,5-diamino-6-hydroxy-2-mercaptopyrimidine (formula 1.16); (iv)2,4-diamino-6-hydroxypyrimidine (formula 1.13); or (v)5-amino-2-chloro-2,3-dihydrothiazolo[4,5]pyrimidine-7-(6H)-one (2.17).36. The method of claim 34, wherein said object is an animal or milk.37-39. (canceled)
 40. A method of selecting a pathogen treatable by (i)the compound defined in claim 1; (ii)4-hydroxy-2,5,6-triaminopyrimidine(formula 1.01); (iii)4,5-diamino-6-hydroxy-2-mercaptopyrimidine (formula 1.16); (iv)2,4-diamino-6-hydroxypyrimidine (formula 1.13); (v)5-amino-2-chloro-2,3-dihydrothiazolo[4,5]pyrimidine-7-(6H)-one (2.17);or (vi) the composition defined in claim 16, said method comprisingdetermining whether said pathogen bears a guanine riboswitch thatcontrols the expression of guaA.
 41. A method of identifying a compoundfor preventing or treating a microbial infection caused by a pathogenbearing a guanine riboswitch that controls the expression of guaA, saidmethod comprising contacting a guanine riboswitch with said compound;determining whether said compound binds to said guanine riboswitch;wherein the binding of said compound to said guanine riboswitch is anindication that said compound is suitable for preventing or treatingsaid microbial infection.
 42. The method of claim 41, wherein saidguanine riboswitch is the guanine xpt riboswitch from Streptococcuspyogenes (STPY-xpt).
 43. The method of claim 41, further comprisingcontacting said guanine riboswitch with guanine or a guanine-likeligand.
 44. The method of claim 43, further comprising determiningwhether said compound may be ribosylated.
 45. A method for preventingthe development of multi-drug resistance of a bacteria in a subject, ortreating a multidrug resistance of a bacteria in the subject said methodcomprising administering a non ribosylable ligand of a guanineriboswitch to the subject, wherein the bacteria bears a guanineriboswitch that controls the expression of guaA.
 46. The method of claim45, wherein said non ribosylable ligand of a guanine riboswitch is: (i)the compound defined in claim 1; (ii) 4-hydroxy-2,5,6-triaminopyrimidine(formula 1.01); (iii) 4,5-diamino-6-hydroxy-2-mercaptopyrimidine(formula 1.16); (iv) 2,4-diamino-6-hydroxypyrimidine (formula 1.13); or(v) 5-amino-2-chloro-2,3-dihydrothiazolo[4,5]pyrimidine-7-(6H)-one(2.17). 47.-49. (canceled)
 50. A kit comprising: (i) the compounddefined in claim 1; (ii) 4-hydroxy-2,5,6-triaminopyrimidine (formula1.01); (iii) 4,5-diamino-6-hydroxy-2-mercaptopyrimidine (formula 1.16);(iv) 2,4-diamino-6-hydroxypyrimidine (formula 1.13); (v)5-amino-2-chloro-2,3-dihydrothiazolo[4,5]pyrimidine-7-(6H)-one (2.17);or (vi) the composition defined in claim 16, and instructions to usesame in the prevention or treatment of a microbial infection.
 51. Amethod for preparing the compound of formula 2.02

said method comprising (a) reacting diethylacetamido malonate withguanidine hydrochloride in the presence of sodium methoxide and methanolto obtain a solid; (b) dissolving the solid of (a) in an aqueoussolution; (c) subjecting the solution of (b) to an acidic precipitationto obtain a 5-formamino-2-amino-4,6-dihydroxypyrimidine precipitate; (d)dissolving the 5-formamino-2-amino-4,6-dihydroxypyrimidine precipitatein an acid; and (e) precipitating the solution of (d) to obtain thecompound of formula 2.02.
 52. The method of claim 51, wherein saidreacting is performed under reflux.
 53. The method of claim 51, whereinsaid acidic precipitation of (b) is performed using a hydrochloric acid(HCl) solution.
 54. The method of claim 53, wherein said HCl solution isa 50% HCl solution.
 55. The method of claim 51, wherein dissolving of(d) is performed using sulfuric acid.
 56. The method of claim 55,wherein said sulfuric acid is 12 M sulfuric acid.
 57. The method ofclaim 51, further comprising washing said solid of (a) with methanol andchloroform.
 58. The method of claim 51, wherein said aqueous solution iswater.
 59. The method of claim 51, wherein said precipitating of (e) isperformed using tetrahydrofuran (THF).
 60. -70.l (canceled)